CIVL407LABMANUAL_20072

Prepared for

Dept. Of Civil Engineering

bySusan C.M. Harper

September 2007

CIVL 407 Lab Manual

Table of ContentsCourse Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Schedule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Station/Drawer Supply List (if supplies are missing, please report) . . . . . 2

First Session: Laboratory Safety and Orientation . . . . . . . . . . . . . . . . . . . . 3

LABORATORY REPORT WRITE UP GUIDELINES . . . . . . . . . . . 5

Laboratory Exercises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Laboratory 1: Solids Determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

5Laboratory 2: COD, DO and BOD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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

1 September 2007 CIVL 407 Lab M anual

1Course Description(Environmental Laboratory Analysis - 1-3-0, 0-0-0)Testing procedures used in water quality studies and in the operation of water andwastewater treatment plants. Prerequisite Chem 151.

Sc h e d u le

Lecture CEME1204: Wednesday 1:00 pm Laboratory CEME1301: 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)

Date 2007 SubjectSep. 4 - 7 No Lab

Sep. 11 - 14 No LabSep. 19 Laboratory Safety & Orientation (~1 hr)

Sep. 25 - 28 #1 Solids determinationOct 2 - 5 #2 Dissolved oxygen, COD, BOD

Oct 9 - 12 No lab - Thanksgiving week - catch upOct. 16 - 19 #3 Bacteriological Examination of waste water

Oct. 23 - 26 #4 ChlorideOct. 30 - Nov.2 #5 Chlorine demand

Nov. 6 - 9 #6 Acidity, alkalinity, hardness, colour, turbidityNov. 13 - 16 No lab - Remembrance day - catch up

Nov. 20- 23 #7a Coagulation Nov. 27 - 30 #7b Coagulation and Softening

Lecturer: Victor Lo, email: [email protected] (604) 822-4880Lab instructor: Susan Harper, email: [email protected] (604) 822-4397

T.A.s: Lab: Margaret Parsotan, email: [email protected]: Kazi Parvez Fattah, email: [email protected]

mailto:[email protected]





Sta t io n /Draw e r Su p p ly Lis t ( i f s u p p lie s a re m is s in g , p le a s ere p o rt )

The following items will be maintained in each stations supply drawer or atstation on bench when required:2 pair safety glasses2 permanent marker pens2 stir bars and a bar retriever1 stir plate2 buret funnels4 30 ml beakers4 50 ml beakers2 100 ml beakers2 150 ml beakers2 250 ml beakers2 1 L plastic beakers2 600 ml plastic beakers2 400 ml plastic beakers2 10 ml grad cylinders2 25 ml grad cylinders2 50 ml grad cylinders2 100 ml grad cylinders1 buret stand1 double buret clamp2 50 ml burets2 25 ml burets2 10 ml burets0-14 pH stripsThermometer2 Stir stick or spatulaFilter funnel (Nalgene magnetic)Tweezers (Millipore or Gelman for membrane filters))

On benches each row:Latex gloves- small, medium and largeNon-latex gloves (keep on reserve for latex sensitive persons only)Filter racks with funnels#4 Whatman filter paper or equivalentVacuum flasks and hoses8 1 L grad cylinders8 3 L plastic beakers


2First Session: Laboratory Safety and Orientation1) NO Food or drink. No sandals or open-toe or heel shoes.2) Personal Protective Equipment: Lab coat (long), eye protection, gloves as

required. 3) Sanitation: antibiotic soap for hand washing, disinfectant for bench surfaces. 4) Hygiene: At times you will be working with sewage. Be aware that handling

your pens, calculators, packs, etc. with contaminated gloves will contaminatethose items. Putting pens in your mouth that may have been on the bench orhandled with gloves could be hazardous. Wearing lab coats out of the lab isforbidden. Please take your labcoat away in a plastic bag.

5) Safety Equipment: Eye Wash Station, Safety Shower.6) WHMIS: Workplace Hazardous Materials Information System is a

government regulation. You must be informed of the hazards of a substancebefore using it. MSDS: is a supplier information sheet that must be availablefor each product and describe the hazards and necessary handling requisites.

7) Spills: Wipe up all spills immediately, wash down and dry the bench. Please askfor assistance if chemicals are spilled.

8) Broken Glass: Be careful not to break glass, but if you do please ask forassistance. DO NOT PUT BROKEN GLASS INTO THE REGULARGARBAGE CONTAINER. If you cut yourself, no matter how minor, pleaseget attendant help.

9) Clean-up: You must leave your work station as you found it - clean and dry.All glassware must be rinsed well (at least 5 times with tap water) and put onpaper towels at your station or on drying racks, if there is room. Do not putlids and small items onto racks that may slip through to the filthy drip tray.Remember to empty and rinse burets as well. Pipettes must be placed with tipspointing up into the pipet basket supplied.

10) Pipettes/ graduated cylinders/ beakers/ flasks:

Volumetric pipettes are the most accurate way to measure volume. They areavailable in 0.5, 1.0, 2.0...up to 10.0 ml, 15, 20, 25, 50 and 100 ml. These should beused whenever standard solutions are measured or when upmost accuracy is required.Most pipettes are calibrated “to deliver” or TD and should never be blown out. They are calibrated by weighing the volume of distilled water that will flow from themby gravity, with the tip against the side of the receiving vessel. A small amount ofliquid always remains in the tip and must not be blown out. Some pipettes may be “to


deliver with Blow-Out”. They are calibrated the same way as TD except that theremaining drop is blown into the receiving vessel. They are usually identified by adouble etched or coloured band at the top of the pipette.

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 isthe most accurate one to select. Some are blow-out, some are not meant to draincompletely - pay attention to the markings.

NEVER mouth pipette. Pipette bulbs or pumps must be used and great care must betaken not to contaminate bulbs. A demonstration will be given. If liquid should entera bulb or pump, please rinse out immediately and set aside to dry. Do not squishbulbs in the direction of another person.

Graduated cylinders: Not as accurate as pipettes, especially at smaller volumes. Theyare useful for measuring samples 20 mls - 1000 mls. Use the size of cylinderCLOSEST TO THE VOLUME YOU WISH to measure for best accuracy. Ie If youwant to measure 20 mls, do not use a 1 L graduated cylinder. Cylinders are available: 5mls, 10 mls, 25 mls, 50 mls, 100 mls, 250 mls, 500 mls, 1, 2 & 4 L

Beakers: are cylindrical in shape with straight sides and flat bottoms; markings arenot accurate at all - merely approximations. Used to hold/transfer approximatevolumes of samples, chemicals or for titrations..

Flasks: Vo lu m e tric flasks are very accurate and should be used when dilutingstandards (together with volumetric pipettes). Erle n m e y e r or conical flask markingsare not accurate. They are containers best suited for “swirling” liquid ie to mixreagents with samples in a colourimetric determination (Lab 4 or 6).

11) Gas/air/vacuum taps: Do not touch these unless instructed to do so. Undetectedgas leaks can be deadly. Even the force of air from the air taps can be dangerousbecause it can contain grit or water.

12) Cup sinks: Please do not use these sinks for the disposal or delivery of liquids.They can be unpredictable in force and are close to eye level. Chemical residues canbe splashed in you face.

13) Stir bars: Please remove from containers before pouring contents down the drain.

14) Glassware/supplies: Each station has a drawer where some glassware and otheruseful tools will be kept. Additional supplies are available in the supply room (aroundthe corner). Pipettes are in drawers indicated. Chemicals will be put out as requiredfor each lab and should be used sparingly, not wastefully as quantities are limited.Make sure that you label beakers and flasks to avoid confusion and waste.

LISTEN AND MAKE NOTE OF VERBAL AND CHALK BOARDINSTRUCTIONS. YOU WILL USUALLY NEED THE INFORMATION

GIVEN TO COMPLETE YOUR LAB WRITE-UPS.


LABORATORY REPORT WRITE UP GUIDELINES

Guidelines for laboratory report write up are also given in the CIVL407 Manual CourseNotes and summarized here. The short report is intended to be a concise yet completelaboratory report whereas the long report is more elaborate. The mark allocation for somesections for both reporting format is indicated in parenthesis in the table below. Please notethat the marking outline is intended only as a guideline to assist in report write-up and thatmarks would also be allocated for overall presentation and clarity of the report.

REPORT ELEMENTS SHORT REPORT

LABS 1 TO 6LONG REPORT

LAB 7

Title page Required Required

Objective Required Required

Materials and Method Details are not required. State that the

procedures were inaccordance with CIVL407 Lab Manual andnote any deviations to

the manual’s procedure.

Required Include a brief description of

the procedure. Sufficientdetail should be provided sothat what was done could beunderstood and the

experiment could be repeatedfrom the procedure reported.

Observations andResults

Required [1] Required [2]

Sample Calculations Required [2] Required [2]

Discussion

Including sources oferror

Brief discussion

required [2]

Required [5]

Conclusions Required Required [2]

Questions and Answers Required [4] Required [6]

Other Students’ Dataand Results

- Required [1]

ReferencesPresented in an

acceptable format.

Required Required

Total Mark [10] [20]


WARNING

You will be handling sewage samples which may contain pathogenic bacteria.Use pipette bulbs, wipe up all spills and wash with disinfectant, keep hands andpencils away from your mouth and wash hands frequently. Do not wear labcoats outside of lab and do not take away from lab unless stored in a plasticbag. Alternatively a place will be provided in the lab to store your coat.

3Laboratory Exercises

Lab o ra to ry 1: So lid s De te rm in a t io n

OBJECT:

-to become familiar with a number of the most common sewagetreatment plant tests and to illustrate some of the difficulties in performingthese tests; to measure sewage treatment plant efficiency in removingresidue.

MATERIALS:-Imhoff cones, samples of raw sewage, sludge, final effluent, oven

muffle furnace, desiccators, balance, evaporating dishes, tin dishes,graduated cylinders, distilled water bottles.

A. SETTLEABLE SOLIDS1. Mix the samples thoroughly and fill individually marked Imhoff cones to the 1 litre

mark.. A 1 L graduated cylinder may be used in place of the Imhoff cone for thesludge sample, if necessary.

2. Settle for 45 minutes, gently dislodge any solids that have clung to the sides using astirring rod, settle for 15 minutes longer, and record the volume of settleable solids. Iflarge pockets of liquid form between the particles of settled matter, subtract anestimated volume from the measured volume of matter. Do not include any surfacefloating material as settled solids.


[Note: Sludge Volume Index is the volume occupied by 1 gram of sludge after 30 minutes ofsettling. This is a different test to the one we are doing using Imhoff cones and should notbe confused. SVI= settled sludge volume (ml/L) x 1000/suspended solids (mg/L)]

B. TOTAL SOLIDS and TOTAL VOLATILE AND FIXED SOLIDS AT 550 Co

1. Obtain the tare weight of a properly prepared porcelain evaporating dish (ie one thathas been washed, dried, pre-fired and desiccated - done for you, prior to session).

2. Mix the sample in the carboy thoroughly, pour about 500 mls (for Part B & Part C)into a beaker and then, immediately after stirring the beaker, rapidly pour sample intoa 50 ml graduated cylinder to approximately equal 35-40 mls (if using 50 ml dishes).[Notes: to allow for rinsing and to avoid spillage when transferring dish to oven don'tpour more than 40 mls].

3. Quickly record the exact volume and pour the sample into the dish.[Note: it may beharder to wash out solids if you allow them to settle before pouring into the dish - sobe quick.] Using very small amounts of distilled water, rinse the cylinder, adding therinsings to the dish.

4. Make sure that you have recorded all the pertinent details: Dish #, tare weight, samplesource and sample volume. Put the dish in the 103-105 C oven for drying. Do not°

write on crucibles as high temperatures during the next steps will burn writing off.5. After drying overnight, the dish will be transferred to a desiccator. Obtain the gross

weight of the dish [Note: it should be room temperature before weighing.]. Subtractthe tare weight to obtain the total solids weight and calculate the mg/L value.

6. The dish containing the dried total solids can now be placed in a muffle furnace or, ifinstructed, on a cart in preparation for staff to do so when all are ready. The sampleswill be fired at 550 C for one hour. The furnace will then be allowed to cool°

significantly before dishes are removed to a desiccator. Once dishes are at roomtemperature they can be re-weighed. The loss in weight represents the volatile solidslost from the originally measured sample volume. Conversely, the solids remainingrepresent the fixed solids. Calculate the mg/L volatile and fixed solids.

C. SUSPENDED SOLIDS - TOTAL AND VOLATILE

1. Obtain the tare weights of three aluminum dishes each containing a glass fibre filter(already pre-washed, dried and fired).

2. Assemble filtering apparatus, position the filter and begin suction (vacuum tap). Wetfilter with a small volume of distilled water to seat it.

3. Using the sample poured out for Part B (for consistency), stir the beaker contents andthen rapidly (so that it doesn't settle) measure a volume of sample into theappropriate graduated cylinder. [Hint: pour out small portions (say 25 mls at a timefor Effl, 10 mls for Raw & 5 mls for sludge) of well-mixed sample and filter entireportion before pouring another portion; when filtering slows significantly do not addmore. Record the total volume filtered.] Rinse the graduated cylinder with smallamounts of distilled water and add to filter. Suggested portions: 100-200 ml of finaleffluent, 25-50 mls of raw sewage and 5-10 ml of sludge u s in g g rad u a te d c y lin d e rs tomeasure - not beakers.

4. Carefully remove filter from filtration apparatus and transfer it back to the aluminumdish. Pinch sides of dish in a bit to protect the filter from oven drafts. Place thealuminum dish into the 103°C oven to dry for at least one hour (or leave drying


overnight).5. Transfer dish to a desiccator, cool and weigh. DO NOT DISCARD! The gain in

weight represents the total suspended solids of the sample. Calculate as the mg/Ltotal suspended solids.

6. The aluminum dish and filter (from #4) must now be fired in a muffle furnace at550°C for at least 30 minutes (or until a constant weight is achieved). For the timebeing, place the dishes on a cart designated by the instructor. Lab personnel willperform the timed-firing after the class. After firing the dishes will be moved to adesiccator. They must be allowed to cool completely before re-weighing them todetermine the loss on ignition or volatile suspended solids. Please return tomorrow toobtain final weight.

7. Calculate the mg/L total volatile solids and total fixed suspended solids.8. From the above determinations it will now be possible to obtain a value for the

dissolved solids present in the sample. Enter all data in the tables on following page.

Calculate the percent solids reduction through the treatment plant.% reduction in settleable matter _________% reduction in total solids _________% reduction in volatile solids _________% reduction in fixed solids _________% reduction in suspended solids _________% reduction volatile susp. solids _________% reduction fixed suspended solids _________% reduction dissolved solids _________

Lab 1 Questions1. What major types of solids are removed in primary treatment? in secondary

treatment?2. If sludge is used in Part A, compute the sludge volume index (SVI). Discuss the

meaning and significance of this index.3. What two techniques can be used to overcome or correct for the loss of material

from filter pads when firing at 550 C?o

4. Your hands may have moisture and natural and/or applied oil/lotion on them. What,if any, affect would there be to the TSS and TVSS results if your bare hands wereused to handle a tin dish a) prior to obtaining the initial tare weight and b) afterobtaining the initial tare weight?

5. A raw sewage sludge goes through an anaerobic digestion process, where the volatilesolids are reduced from 65% to 40%. If all of the volatile solids reduced is given offas gas and if the specific gravity of the volatile solids is 1.3 and fixed solids 2.50, whatis the percentage reduction in solids.

6. Classify each of the following into its proper solids category(s) i.e. dissolved,suspended, volatile, fixed (use more than one adjective to describe each substance.)a) a bacteriumb) sodium chloridec) sugard) clay


DATA AND RESULTS

Raw Sewage Aerobic Sludge Final EffluentA. Settleable Matter After 1 hour (mL/L)

B. Total Solids Dish marking (do not use ink - use permanentmark on dish) Sample volume (mL)

Gross weight - oven dry (G)

Dish tare weight (G)

Total solids (G)

Total solids (mg/L)

Gross weight - fired (G)

Loss on ignition (G)

Volatile solids (mg/L)

Fixed solids (mg/L)

Percentage fixed residue

C. Suspended Solids Tin dish marking (do not use ink - use embossedmark) Sample volume (ml)

Gross weight - oven dry (G)

Tin dish with filter tare weight (G)

Total suspended solids (G)

Total suspended solids (mg/L)

Gross weight - fired (G)

Loss on ignition (G)

Volatile suspended solids (mg/L)

Fixed suspended solids (mg/L)

Dissolved solids (mg/L)


WARNING

You will be using concentrated acid and other corrosive and toxicchemicals, as well as handling samples probably containing pathogenicbacteria. Wipe up all spills immediately, wash/rinse bench tops withwater/disinfectant. NEVER pour water into acid. Keep fingers, pencils,etc out of your mouth. Wash hands frequently with soap. Always useand store pipettes with the top higher than the tip, otherwise reagentswill run to the bulb end and make pipetting difficult and contaminationlikely.

5Lab o ra to ry 2: CO D, DO an d B O D

OBJECT: -to familiarize you with the commonest tests run for assessingtreatment plant efficiency and pollution loading to receivingwaters; to illustrate the shortcomings of these tests; and todemonstrate the use of dissolved oxygen probes.

I. COD - Closed Reflux, Colourimetric Method

NOTE: Open reflux method is the most accurate means of determining CODbecause a large sample size is used (20 mls). The closed reflux method is moreeconomical but because a small sample volume (2 mls) is used, homogenization ofsamples containing suspended solids may be necessary in order to get arepresentative sample and to improve reproducibility. Digests from either methodcan be titrated or read colourimetrically.

Materials: Raw sewage and final effluent samples, vials with pre-measured reagents,Hach block digester, spectrophotometer, potassium hydrogen phthalatestandards: 800, 400, 200, 100 and 50 mg/L as COD, pipettes, etc.


Procedure:1. At your station you will find four vials with pre-measured solutions containing

potassium dichromate and sulfuric acid (prepared according to standard methods,except without mercuric sulfate). Put identifying labels on each tube ie your initials, Raw Sewage (Infl) and/or Effluent (Effl) in the upper 1/4 of the tube usingpermanent markers.

2. Pipette 2 mls of sample (using volumetric pipettes unless particulate is present, inwhich case, use wide mouth graduated pipettes) in duplicate, into the appropriatetubes and screw cap on snugly. If sample is known to be outside the standard range(above 800 mg/L), a smaller aliquot (making volume up to 2 mls with distilled water)or 2 mls of a pre-diluted sample can be used. Make sure sample is well mixed withthe acid contents of the vial. [Note: it should look homogenous and be careful it willbe very hot!]

3. Take vials to fumehood and place in block digester - noting the location of yourwell-labelled, well-mixed samples in the 25-position digester.

4. Standards have been digested for you, before the lab, and are at thespectrophotometer. Allow samples to digest for 30 minutes (normally this would be2 hours). Remove the vials and allow them to cool to room temperature (use coldwater to speed up if necessary).

5. The instructor will demonstrate the use of the spectrophotometer, which is set at600 nm to read absorbance of light by the sample. The vials should be clean and dryon the outside. Read your samples and the set of COD standards: 800, 400, 200, 100,

250 and 0 COD as mg O /L.6. Prepare calibration curve, absorbance versus concentration and calculate the COD in

samples. If less than two mls of sample or diluted sample was used, a volumetriccorrection must be made to the value obtained from the curve.

II. DISSOLVED OXYGEN (DO)

Materials: Raw sewage and final effluent samples, dilution water, alkaline-iodide-azidereagent, manganous sulfate, concentrated sulphuric acid, starch indicator,0.025 N sodium thiosulphate, thermometers, BOD bottles, 10-ml graduatedpipettes, burette stand with burettes, 250-ml graduated cylinder, 500-mlErlenmeyer flasks.

Note: You are going to measure DO on four different liquids using two different methods.The four liquids are cold tap water, aerated dilution water, raw sewage and finaleffluent.

You will use a chemical titration (Winkler - the azide modification of the iodometricmethod) and a dissolved oxygen probe and meter. The probe has been previouslycalibrated. DO NOT ADJUST THE SETTINGS ON THE METER.


A. DISSOLVED OXYGEN PROBENOTE: DO NOT add any reagents to the samples used for the probe method.

1. Fill four BOD bottles with the four samples (cold tap water, aerated dilution water,raw sewage or influent and final effluent), right to the top and place stopper onbottle. (Fill carefully, taking care not to entrain additional air. For the cold tap watersample, submerge the hose in the bottle and allow the water to overflow for 15-30seconds).

2. Determine DO, using the DO probe and meter, for each of the four samples. Instructions on the use of the probe will be given in the lab. Record the temperatureof each sample using the DO meter or a thermometer. (Note: DO metertemperature is usually not accurate.) You can save these bottles for B.

B. WINKLER TITRATION (Azide modification)NOTE: Use only the droppers attached to the reagent bottles for dispensing into samples.Do not mix droppers and do not allow tap water into the reagent bottles.

1. Fill a BOD bottle with each of the four samples (or use ones saved from A ).Stopper the bottles without trapping any air bubbles.

2. Place the bottles in the sink and one at a time remove the stopper, add 1 (one) mlof manganous sulfate reagent using plastic dropper provided (keeping the droppertip just below the surface) and quickly replace the lid.

3. To the same bottles, add 1 ml of alkaline-iodide-azide solution the same way, againquickly replacing the lid.

4. Take each stoppered bottle, tip the liquid out of the space around the stopper(caution -it may be corrosive) and invert several times, as demonstrated, tothoroughly mix contents. Set back in sink.

5. Allow the floc to settle to about 1/3 the bottle volume, invert several times again andallow to settle again. When the floc has settled the second time to about 1/3 bottle

2 4volume, remove the stopper, add 1 ml of concentrated H SO and quickly re-stopper. Tip out the liquid from the area around the stopper and invert several timesto mix thoroughly. The chemical floc should completely dissolve. (Note: biologicalsolids originally present in the sample will not dissolve.)

6. Transfer 201 ml of each bottle (do one at a time) to a 500 ml conical flask. Use thespecially marked volumetric flask to dispense the 201 ml.

2 2 37. Titrate this volume with 0.025 N Na S O until only a faint yellow or "straw"colour remains. Note: if solution already seems pale yellow add starch rightaway. Add enough starch solution to give a strong blue colour (one dropper full)and continue to titrate, dropwise, until the first complete disappearance of the bluecolour. Neglect any reappearance of the blue colour. Record the volume of titre.(Hint: if you measured very low DO level with the meter, then the same sample willrequire little or no titre and may not turn blue when starch is added - think about it!)

8. Complete all four titrations. The DO concentration in mg/L is equal to the numberof ml of titre as long as 201 ml is titrated.

9. Determine the percent saturation for each sample.


Dissolved Oxygen Data

Sample/Data Raw Sewage Final Effluent Tap Water Dil’n Water

Bottle #

Sample Volume (mls)

Initial Buret reading

Final Buret reading

Net Titre (mls)

DO conc. (mg/L)

DO conc. (Probe)

Temp ( C) (thermometer)o

Saturation concentration

Percent saturation

DETERMINING PERCENT SATURATION THE "QUICK AND EASY" METHOD

For a quick and easy determination of the percent saturation value for dissolved oxygen at a given

temperature, use the saturation chart above. 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. The percent saturation is the value where the line intercepts the saturation scale. 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.


III. BIOCHEMICAL OXYGEN DEMAND

5Note: Because of the logistics of organization some groups may have 6 day BODinstead of the usual 5 day. Be sure to note this in your lab write-up.

Materials: Raw sewage and final effluent samples, dilution water, BOD bottles, 10-mlgraduated pipettes, graduated cylinders. Dissolved oxygen meter and probe.

1. Using the volumes and samples provided by the instructor, carefully measure thewaste water samples into BOD bottles. Two dilutions will be made for each sample and each dilution will be in duplicate for a total of eight bottles (plus one bottle forthe blank mentioned in Step 4). Be sure to record the bottle numbers and samplevolumes on the following table - do not write on the bottles and do not use thenumbers on the lids.

3. Carefully top up the BOD bottles with DILUTION WATER, to bring the levelsabove the ground glass neck of the bottle. Try not to aerate the contents by keepingthe filling tube below the surface of the liquid in the bottle or carefully running thewater down the side of the bottle. Measure the Initial DO using a calibrated probeand meter.

4. In addition, fill one BOD bottle with DILUTION WATER alone. Because you wantto be sure that the dilution water has no BOD, be careful not to contaminate it withsample. ie Rinse the filling tube if it has been in the sample. The blank is used tocheck that the dilution water has negligible BOD. Measure the Initial DO of theblank using the calibrated probe and meter. There should be negligible BOD in theDilution water, but if there is a significant BOD then it will have to be subtractedbefore the dilution factor is applied in the calculation. If the dilution water BOD isgreater than 1 ppm, it may indicate that probe may not have been calibrated properlyor the dilution water had been contaminated.

5. Stopper the BOD bottles, being sure to exclude any air bubbles that might betrapped under the stopper by adding dilution water. All bottles should have liquid inthe well around the stopper - if not add a some dilution water (or distilled water) tothe well. Place plastic caps over all bottles. These caps are designed to prevent theevaporation of the liquid around the stopper, thereby maintaining a water seal foreach bottle.

6. Place all bottle sets in the 20 C incubator for five days (or 6 for Tues. groups). Aftero

five (or six)days, remove bottles and measure the DO, preferably using the samemeter and probe used to get IDO reading. Calculate the 5-day (or 6-day ifunavoidable) BOD, showing sample calculations. Note: Each group should have 9BOD bottles in the incubator.


BIOCHEMICAL OXYGEN DEMAND (BOD) DATA

Sample source: ________________________________

Raw SewageBottle 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 waterBottle number (not lid #) _____Sample volume (ml) _____Initial DO (mg/L) _____Final DO (mg/L) _____

BOD CalculationWhen dilution water is not seeded (our water is not seeded for this exercise):

When seeded water is used:

1where: D = DO of diluted sample immediately after preparation, mg/L2D = DO of diluted sample after 5 d incubation at 20 C,o

mg/L.P = decimal volumetric fraction of sample used

1B DO of seed control before incubation, mg/L=

2B = DO of seed control after incubation, mg/L, andf = ratio of seed water in diluted sample to seed in seed

control= (%seed in diluted sample) / (%seed in seedcontrol).


Summary

Raw Sewage, Diln. 1 ________ ________Raw Sewage, Diln. 2 ________ ________ Average ________

Final Effluent, Diln. 1 ________ ________Final Effluent, Diln. 2 ________ ________ Average ________

Percentage BOD reduction through treatment plant ___________

Lab 2 Questions

1) At what relative times should influent and effluent samples be taken from a sewagetreatment plant in order to determine true % removal efficiency?

2) Give two reasons why samples for dissolved oxygen should be “fixed” in the field, ifat all possible.

3) Calculate theoretical oxygen demand for 300 mg/L of methyl alcohol.

4) Show on a graph the approximate dissolved oxygen sag curves in a river receiving

5two wastes. Both wastes have equal BOD temperature and volume. One waste has aK value of 0.17, the other 0.25.

55. If an industrial waste has a COD of 450 mg/L, what would you expect its I) BOD

Land ii) BOD to be.

6) What interference does the Azide Modification of the Winkler Dissolved Oxygenmethod overcome and why is it the most common method used for domesticsewage?

7) Why is a blank titrated in the COD test? (Refer to standard methods, open reflux,titrimetric method.)

8) Why is your COD result different from the BOD result?


WARNING

As you are handling and pipetting samples probably containing pathogenic organismsplease observe the following rules: Wear gloves, lab coats, eye protection; DO NOTMOUTH PIPETTE; wipe up all spills and disinfect the area of the spill. Keep hands,pencils, etc., away from your mouth. Wipe lab benches with disinfectant before andafter each lab. Wash hands with soap before leaving. Do not wear lab coat out of lab- leave it here or take away in a plastic bag to launder.

Lab o ra to ry 3: B a c te rio lo g ic a l Exam in a t io n o f Se w ag e

OBJECT:-to demonstrate different methods for the determination of the coliform

group of organisms and to acquaint you with bacteriological procedures. Studentswill do only sections marked ?

MATERIALS:Multiple tube method: dilution tubes, lauryl tryptose broth tubes, Brilliant GreenLactose Broth tubes, EC tubes and/or Hach A1 tubes. Demonstration only.Membrane filter method: dilution tubes, sterile pipettes, sterile dilution water,filtering apparatus, membrane filters, LES Endo agar plates, 35 C incubator.o

Heterotrophic plate count: dilution tubes, sterile pipets, agar tubes, plates, 35 Co

incubator, Quebec colony counter.HACH P/A Broth with MUG. Demonstration only

SAMPLE: You will do either a raw sewage sample or a final effluent sample, notboth. Get raw data from another group and include in your report.

Fiv e d i f f e re n t p ro c e d u re s w i ll b e s tu d ie d - b e c a u s e o f lo g i s t i c s i t i s 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 ri t t e n . T h e re f o re , y o u w i ll d o th o s e m a rke d b y ? only


A. Multiple-tube Fermentation or Most Probable Number: Serial dilutions of sample are incubated in lauryl tryptose broth at 35 C for 48 hours (swirl after 24o

hrs). Gas formation indicates the possible presence of coliform organisms. Another series of tubesare inoculated from these positive lauryl tryptose tubes. Brilliant green lactose bile (BGLB) tubesare inoculated and incubated at 35 C for 48 hours (confirmed test for total coliforms) and EC tubeso

are inoculated and incubated at 44.7 C for 24 hours (confirmed test for faecal coliforms). Bacterialo

numbers are calculated from the numbers of positive tubes. Hach’s Most Probable NumberMethod 8368, A-1 Medium, is USEPA-accepted for testing non-potable waters. It is used as afaster alternative to the LTB/EC procedure for enumerating faecal coliforms in water. Note: thesemethods will be demonstrated but data must be recorded.

? B. Membrane Filter Technique: Serial dilutions of sample are collected on membranefilters and placed on M-Endo media. Colonies that are pink to dark red with a golden green metallicsheen are counted after incubation 20-22 hrs at 35 C.o

? C. Heterotrophic Plate Count - Pour Plate Method: Serial dilutions of samples mixedwith agar and incubated for 48 hrs at 35 C. The number of colonies counted directly.o

D. Presence/Absence Testing: HACH technique to simultaneously screen for total coliformand E.coli, using a ready-to-use P/A Broth with MUG. MUG produces a fluorogenic productwhen hydrolyzed by an enzyme specific to E.coli. The broth will turn yellow indicating totalcoliform and fluoresce in the presence of E.COLI in 4 to 24 hours. Demonstration.

E. Microscope Slides (Completed Phase of Multiple-Tube Fermentation): Singlecolonies arising from single cells are prepared and stained for microscope examination. (Singlecolonies are obtained by streaking LES Endo agar plates with an inoculum from positive BGLB orEC broth tubes and incubating for 24 hours.)

Methods:? Recording BGLB, EC or A-1 tubes positives: 1. All tubes have been examined for gas production. Formation of gas in any amount in the

inverted vial of the BGLB broth fermentation tube at any time within 48 hr ±3 hrconstitutes a positive confirmed phase. Calculate the MPN value from the number ofpositives using the Table 1. Express as Total coliforms. Results will be provided for thedemonstration set.

2. Consider positive EC broths incubated at the elevated temperature (44.5 C) as ao

positive completed test response for both Total and faecal coliform. Parallel positiveBGLB broth cultures with negative EC broth cultures indicate the presence of non-faecal coliforms.

3. Gas-positive Hach A-1 tubes indicate the presence of faecal coliform. Use Table 2on page 27.

Completed Phase LES Endo agar Plates: A p la t e w ill b e a v a ila b le to lo o k a t .1. Using aseptic technique, as demonstrated, streak one LES Endo agar plate from each

tube of BGLB broth showing gas from the highest dilution and the second highestdilution. Streak plates in a manner to insure presence of some discrete colonies.


Flame loop between second and third quadrants to improve colony isolation.2. Incubate plates (inverted) at 35 C for 24 ± 2 hr. Prepare Gram Stain slides foro

examination under a microscope..

?B. Membrane Filter Technique -Total Coliform1. Dilution tubes. If you haven’t already, prepare one set of six dilution tubes following

the instructions given under Multiple Tube Fermentation.2. Filtering. The filtration apparatus has been sterilized by autoclaving. Mount

apparatus on suction flask with valve sideways to block suction. Using sterileforceps, place a sterile membrane filter (grid side up) onto the filtration apparatus.Pipette about 10 ml of sterile dilution water over the surface of the filter with thesuction valve still closed. Pour the full volume of dilution tube # 6 onto the filter,open the valve slowly and allow all the liquid to be pulled through. Close valve andrinse the funnel with three aliquots (portions) of sterile dilution water opening andclosing valve after each aliquot.

3. When filtering is complete and the valve is closed, remove filter using flame-sterilized forceps (allow forceps to cool or they will damage the filter). Place the filteron a small LES Endo agar plate carefully, from one edge, with a motion thatexcludes air as it comes in contact with the agar. Do not push down on the filterexcept on the outside edge and use only sterile forceps - applying gentle pressure.

4. Repeat for the rest of the dilution tubes to # 2. Incubate inverted Petrie dishes for20-22 hr at 35 C. Be sure dishes are labelled with your name and dilutions.o

5. Counting. After 20-22 hr, choose appropriate dilutions for counting. Use dishes with20 to 80 coliform colonies and not more than 200 of all types. The typical coliformcolony has a pink to dark-red colour with a golden green metallic surface sheen. Thesheen area may vary in size from a small pinhead to complete coverage of the colonysurface. The sheen area may vary in size from a small pinhead to complete coverageof the colony surface. Atypical coliform colonies can be dark red or nucleatedwithout sheen. Colonies that lack sheen may be pink, red, white, or colourless andare considered to be non-coliforms. The total count of colonies (coliform and non-coliform) on Endo-type medium has no consistent relationship to the total numberof bacteria present in the original sample. A high count of non-coliform coloniesmay interfere with the maximum development of coliforms. Verification of bothtypes of colonies is advisable as some typical sheen colonies may be noncoliform andsome atypical may be coliform. Verification is usually done by a test for lactosefermentation. Gram staining technique can be also be used. In general, coliformbacteria are Gram-negative (pink) rods (sometimes coccus-like).

-Note: “typical” colonies count as coliforms in this procedure, therefore the results tableshould specify #coliform colonies/100 ml.

?C. Heterotrophic Plate Count - Pour Plate Method1. Dilution tubes. Prepare one set of six dilution tubes as described in Section A.1.2. Inoculating. Using a sterile pipette, transfer 1 ml of sample from dilution tube # 6

(most dilute for Raw sewage) or #5 (for Effluent) to an empty (large) Petrie dish,removing the cover just enough to insert pipette tip and dispense sample, thenimmediately replace the cover.


3. Repeat with dilutions 5 to 2 (for Raw) and 4-1 (for Effluent).4. Take 5 tubes of melted agar from the incubator. Let them cool down from the 60C

that they are kept at but not enough to solidify. Test temperature on your wrist - toohot for a baby - too hot for a bacterium. Slide the cover of a Petrie dish back just farenough to pour the agar, pour and replace cover. Gently swirl the dish, on the bench,to mix the sample with the agar. Repeat for the rest of the dilutions. Work quicklyor the agar will solidify in the tube before you get a chance to pour it.

5. Allow the agar to solidify in the dish before inverting, marking (on edge of dish so asnot to impede counting) and incubating for 48 hr at 35 C.o

6. Counting. After 48 hr incubation, select the appropriate dilution to count. Ideally,use the plate(s) that will give from 30 to 300 colonies/plate. The aim of using severaldilutions is to have at least one dilution giving colony counts between these limits.Use the Quebec colony counter to aid in counting and a grease pencil to mark offcolonies counted. A hand counter is also available. Compute bacterial density andreport as “colony-forming units” (CFU) per ml. Do not report as “too numerous tocount” if all plates exceed 300 colonies. Instead, if fewer than 10/cm , count2

colonies in 13 squares (of the colony counter) of the most dilute plate havingrepresentative colony distribution. Preferably select seven consecutive squareshorizontally and six consecutive squares vertically. Multiply the number by 5 tocompute estimated colonies per plate (66 cm ). If more than 10/cm , count four2 2

representative squares, average and multiply by 57 (for plastic Petrie dish). Ifnecessary, refer to Standard Methods for more instruction.

D. Presence/Absence Testing - d e m o n s tra t io n - s ta rt e d ah e ad o f la b1. Add 100 mls of sample directly to pre-measured medium provided.2. Incubate at 35 C for 24-48 hours.o

?3. Examine for yellow colour (total coliforms) and fluorescence (E.coli).?4. Record results of previously prepared test.

E. Microscope Slides: 1. Slide preparation: With a flame sterilized inoculating loop, place 1 loopful of sterile

dilution water in the centre of a slide. Resterilise the loop and remove one colonyfrom the surface of the Les Endo streaked plate (prepared previously). Spread thecolony in the drop of water so that you get a uniform dispersion over an area aboutthe size of a dime. Do this for a typical and an atypical colony. Label the slide usinga grease pencil (T or A).

2. Slide staining.a. Air dry the slide high above the Bunsen burner and fix by passing slide briefly

through a flame.b. Flood slide with crystal violet solution and allow to act for 30 seconds.c. Wash off stain with water and then with iodine solution (Lugol’s).d. Allow iodine to act for 30 seconds.e. Drain off excess iodine and wash freely with water and then alcohol-acetone

decolourising solution. Flood the slide with fresh alcohol-acetone solution toact for 30 seconds.

f. Wash slide in tap water.


g. Apply fuchsin or safranin (counter)stain for 30 seconds.h. Wash in water, blot and then dry in air high above the Bunsen burner (so as

not to burn away your efforts).

?3. Microscopic examination. a. Examine the prepared slides - do not turn knobs except the positioning ones.b. In general, coliform are Gram-negative (pink), nonsporing, usually motile,

short, plump rods, sometimes coccus-like and 0.5 - 3 µm in size. Cells occursingly, in pairs, or in short chains. Note: in the staining process: beforedecolourisation all organisms appear Gram positive (blue), afterdecolourisation Gram negative organisms are no longer visible. Afterapplication of the counterstain, Gram negative organisms are visualized(pink).

c. Description of bacteria: Describe what you see on the plates: colonialmorphology of each type of colony seen ie surface (shiny, dull, metallic,rough, smooth, concave), transparency, colour, odour; and on the slide:typical or atypical, gram positive or negative, cocci or rods, length to widthratio, cell groupings (single, pairs chains).

Schedule Summary

Day 1. - Record positives in all Multiple Tube Fermentation inoculations: LTB, BGLB and EC andAI tubes - counts will be provided on the board.- Prepare dilutions for Heterotrophic Plate Count and Membrane Filter technique.- Prepare and incubate Heterotrophic (Pour) Plates using dilutions above.- Membrane filter prepared dilutions, place on LES Endo agar dishes and incubate.- Record Presence/absence testing results of Hach Prepared Media bottles.- Examine prepared slides.

Day 2.- Come in and count colonies on LES Endo Membrane Filter dishes.

Day 3.- Come in and count Heterotrophic Plate colonies after 48 hours. Plates ready on a weekendwill be put in cold room until Monday.


Bacteriological Examination DataMultiple Tube Fermentation : indicate the number of positives for each dilution

Tube # 1 2 3 4 5 6

Mls of sample/dilntube

Infl

Effl

LTB 24 hr (+ or -) Infl

Effl

BGLB 48 hr (+ or-)

Infl

Effl

EC 24 hr (+ or -) Infl

Effl

AI 24 hr (+ or -) Infl

Effl

From MPN Index table find MPN Index per 100 ml. Multiply the MPN index by the dilution factor fromthe series used when dilutions other than 1/1, 1/ 10 and 1/100 are used..

Heterotrophic Plate Count - pour plate method, 35 C/48 hro

Tube # 1 2 3 4 5 6

Mls of inoculum Infl

Effl

Plate count Infl

Effl

*CFU/ml Infl

Effl

*CFU= colony-forming units

Membrane Filter Technique

Tube # 1 2 3 4 5 6

Dilution (mls) Infl

Effl

Coliform Colonies Infl

Effl

#Colonies/100 mls Infl

Effl


Discussion:

-sources of error?-compare MPN, MF results. Do they agree? Why/why not?-compare MPN/MF with Heterotrophic Plate Count. Do they agree? Why/why not? Whatis expected? What is a heterotroph?-were the reductions through the sewage treatment plant as expected?

Additional notes

Presence/Absence (P/A) Testing:

Media chosen may be Presence/Absence broth (P/A) or Lauryl Tryptose broth with

Bromcresol Purple broth (LT/BCP) to indicate acid formation. Both media meet USEPA

guidelines for testing of total coliforms in drinking water. They are in concentrated form

and merely require the addition of 100 ml of sample (or diluted sample) to the media and

incubation. If zero total coliform is the maximum contamination goal, it is not necessary to

enumerate.

With MUG reagent added to P/A Broth, you can simultaneously detect total coliforms and

E.coli. A long-wave UV light is required to detect fluorescence. MUG (4-

methylumbelliferyl-$-D-glucuronide) can be used to detect E.coli because it produces a

fluorogenic product when hydrolyzed by glucuronidase, an enzyme specific to E.coli. E.coli

will fluoresce as early as 4-24 hours.

Estimation of Bacterial Density - Most Probably Number (MPN)

When more than three dilutions are used in a decimal series of dilutions, use the results

from only three of these in computing the MPN. To select the three dilutions to be used in

determining the MPN index, 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. Use the results at these three volumes in computing the MPN index. In the

examples given below, the significant dilution results are in boldface. The number in the

numerator represents positive tubes; that in the denominator, the total tubes planted; the

combination of positives simply represents the total number of positive tubes per dilution:

Example 1

ml

0.1

ml

0.01

ml

0.001

ml

Combination

of positives

MPN Index

/100 ml

a 5/5 5/5 2/5 0/5 5-2-0 5000

b 5/5 4/5 2/5 0/5 5-4-2 2200

c 0/5 1/5 0/5 0/5 0-1-0 20


Table 1 - MPN Index and 95% Confidence Limits for Various Combinations of PositiveResults when 5 Tubes are used per Dilution (10 mL, 1.0 mL, 0.1 mL)

Comb.OfPositives

MPN Index/

100 ml

95% Confidence Limits

Lower UpperComb. Of

Positives

MPN

Index/

100 ml

95% Confidence Limits

Upper Lower

0-0-0 <2 - - 4-2-0 22 9 56

0-0-1 2 1 1 4-2-1 26 12 65

0-1-0 2 1 10 4-3-0 27 12 67

0-2-0 4 1 13 4-3-1 33 15 77

1-0-0 2 1 11 4-4-0 34 16 80

1-0-1 4 1 15 5-0-0 23 9 86

1-1-0 4 1 15 5-0-1 30 10 110

1-1-1 6 2 18 5-0-2 40 20 140

1-2-0 6 2 18 5-1-0 30 10 120

2-0-0 4 1 17 5-1-1 50 20 150

2-0-1 7 2 20 5-1-2 60 30 180

2-1-1 9 3 24 5-2-0 50 20 170

2-2-0 9 3 25 5-2-1 70 30 210

2-3-0 12 5 29 5-2-2 90 40 250

3-0-0 8 3 24 5-3-0 80 30 250

3-0-1 11 4 29 5-3-1 110 40 300

3-1-0 11 4 29 5-3-2 140 60 360

3-1-1 14 6 35 5-3-3 170 80 410

3-2-0 14 6 35 5-4-0 130 50 390

3-2-1 17 7 40 5-4-1 170 70 480

4-0-0 13 5 38 5-4-2 220 100 580

4-0-1 17 7 45 5-4-3 280 120 690

4-1-0 17 7 46 5-4-4 350 160 820

4-1-1 21 9 55 5-5-0 240 100 940

4-1-2 26 12 63 5-5-1 300 100 1300

5-5-2 500 200 2000

5-5-3 900 300 2900

5-5-4 1600 600 5300

5-5-5 $1600 -— ----


Hach - MPN Table 2


Further information can be found in the laboratory library (do not remove withoutpermission):

Standard Methods: -Part 9000 Microbiological Examination

HACH publications:-Catalogue listing testing methods with descriptions.-The Use of Indicator Organisms to Assess Public Water Safety- http://www.hach.com/ ¸Information Central/Learning

Library/Microbiological Testing

ASTM: -Bacterial Indicators/ Health Hazards Associated with Water(STP635)

D.D. Mara: -Bacteriology for Sanitary Engineers

Microbiology 321: -Manual of Microbiological Techniques

Website:http://www.microbelibrary.org/microbelibrary/files/ccImages/Articleimages/keen/Gramstainkeen.htm

Lab 5 Questions:

1) Compare the advantages and disadvantages of the multiple tube fermentation andmembrane filter techniques.

2) What are the important pathogens transmitted by water?

3) How is quality control achieved in microbiological analysis.

http://www.hach.com/


W A R N I N G

You will be using concentrated acid and other very dangerous chemicals.Mercuric thiocyanate is very toxic. Use personal protective equipment: gloves,goggles or glasses and lab coats. If chemicals are spilled alert instructors,immediately.

Lab o ra to ry 4: Ch lo rid e De te rm in a t io n

OBJECT:

-to acquaint you with the use of specific ion electrodes, known addition and calibrationtechniques and colourimetric and potentiometric titrations; to illustrate the differences inoperation and accuracy between instrumental methods and wet chemical methods.

MATERIALS:

-millivolt meter, chloride specific ion electrode, reference electrode (double junction),magnetic stirrer, beakers, buffer, standards, semi-log graph paper; spectrophotometer, 125ml Erlenmeyer flasks, graduated cylinders; pH meter with double-junction electrode and

3silver billet electrode, burettes, conc. HNO , standard chloride solution (0.5 mg/L),

3 3 2standard AgNO solution, volumetric pipettes; standard Hg(NO ) , indicator-acidifier

3reagent, indicator, NaHCO , beakers; sample.

Note and consider carefully: Different methods of analysis have different optimumranges. You will be told the approximate concentration in the sample and it is up to you tomake sure that the sample will fit within the specified ranges. That means you will probablyhave to dilute the sample by the most accurate means available. Hint: optimum ranges willcoincide with the standards provided or will be indicated within the pertinent section.


I. CHLORIDE - SPECIFIC ION ELECTRODE

Part One: Preparation and reading standards and samples

1. Standards of 1000, 100 and 10 ppm (mg/L) have been prepared for you and are atthe meter. Unless already done by a previous group, pour out 100 mls of eachstandard into separate, labelled beakers. Add 2 mls of IONIC STRENGTHADJUSTMENT (ISA) buffer to each using the plastic dropper (filled to the mark).

2. Unless already done by a previous group (values written on the board), place thelowest standard on the stirrer and lower the electrodes into it. Turn on stirrer andwhile stirring record the millivolt (mv) reading when the reading appears to bestable. As probe tends to drift for some time it may be expedient to select a“standard time to read” (i.e. 2 minutes) and use it for all standards and samples.Repeat for rest of standards, rinsing and blotting dry the electrodes between each.

3. Measure 100 mls of sample, added 2 mls of ISA, stir and obtain a reading for it. 4. Using semi-log paper or software equivalent, plot the millivolt readings (linear axis)

against the concentration of the standards (log axis) to produce a calibration curve.Determine the concentration of chloride in the sample.

5. Method of Standard Addition :Add 5 mls of the stock (4 mg/ml) chloride solution

s 1(V ) to the sample measured in Step 3 (E ) and take a new reading after value is

2stable (E ).6. Calculate the chloride concentration, where:

1E = potential of sample (mV)

2E = potential of sample plus stock addition

2 1E = potential change = E - E

oV = volume of sample

sV = volume of stock solution addedA = mg Cl added to the sample-

S = "slope" = change in potential over 10 fold change in concentration ofstandard (use data from Step 2).

mg Cl in sample = -

mg/L Cl = ____________________-


II. CHLORIDE - BY COLOURIMETRIC METHOD

The principle of this method is based on the following formula:

2 22Cl + Hg(SCN) º HgCl + 2SCN- -

SCN + Fe º Fe(SCN) (Reddish brown)- +++ ++

1. Using a 25 ml graduated cylinder, measure 25 mls of “zero” standard or “blank”(halide-free distilled water), the three standards (2.0, 4.0 and 8.0 ppm), and thesample (diluted if necessary) into separate and labelled 125 ml Erlenmeyer flasks (5in total).

2. Marking the time (T=0), add 5 ml of the “combined reagent” (containing ferric alumand mercuric thiocyanate solutions) to the blank first, using the dispenser provided.(Use extreme caution as this is a corrosive & toxic chemical.) Mix thoroughly bygently swirling the flask.

3. Wait two or three minutes and then add the reagent to the first (lowest) standard;after a few more minutes, add the reagent to the second standard and so on for therest of the standards and sample (which may need to have been diluted). [Note: thisdelay between additions is to allow you time to measure absorbance of each at the10 minute time required - they can’t all be at 10 minutes at once.]

4. After 10 minutes has passed (since the combined reagent was added to the blank),use a plastic dropper to transfer blank into a spectrophotometer cuvette (do not fillmore than 3/4 ), discard and refill at least twice to rinse cuvette. Insert cuvette intothe spectrophotometer (wavelength must be set at 463 nm) and press “zero” or “refset” (depending on model) to make the meter read zero. (Alternatively, zeroinstrument on distilled water and obtain an absorbance value for the blank, whichthen must be subtracted from the standards and sample.)

5. Every two or three minutes thereafter you will rinse the cuvette with the nextstandard or sample, fill, insert in the spectrometer and record the absorbancedisplayed on the meter. (No further adjustment is required after the blank or distilledwater has been set to zero.)

6. Prepare a calibration curve by plotting the absorbance readings versus theconcentration of chloride in the standards. Determine the chloride concentration inthe sample from the calibration curve. If sample is above the highest standard, itshould be rerun from the start after diluting.

III. MERCURIC NITRATE METHOD FOR CHLORIDE DETERMINATION

The principle of this method is as follows: Cl ions in the sample combine with added-

mercuric ions forming a soluble but virtually undissociate-able complex. After all Cl ions-

have been bound, the excess mercuric ions combine with diphenylcarbazone (DPC) toproduce a violet colour (the end-point).

2Hg + 2Cl º HgCl and Hg (excess) + DPC º violet colour++ - ++

1. Pour 100 ml of sample (or a portion diluted such that the concentration is less than100 mg/L) into a 250 ml beaker..

2. Add 1 ml (use pipette to measure accurately) of indicator-acidifier reagent to sample.The colour of the solution should be green-blue at this point. (Light green indicatesa pH of less than 2.0; pure blue, a pH of greater than 3.8. For most samples, the 1 mlof the indicator-acidifier reagent will adjust the pH to 2.5 ± 0.1.

3 23. Titrate with 0.014N Hg(NO ) to a definite purple end-point. Near the end-point,the solution should be green-blue and will turn to pure blue (no green) within a few


drops of the purple end-point.4. Determine the blank by titrating 100 ml of distilled water containing 10 mg of

3NaHCO which serves to provide some alkalinity to the blank (to make it similar tothe sample) and provides a correction for alkalinity and reagents - if one is necessary.Don’t forget to add the indicator. The titre from this step must be subtracted fromthe titre for the sample.

5. Calculate the chloride concentration:

where: A = ml titrant for sampleB = ml titrant for blankN = normality of titrant

IV. CHLORIDE BY POTENTIOMETRIC TITRATION

Note: For this part form two groups comprised of 1 member from each station. The firstgroup will do the standard (part one) in duplicate or triplicate depending on the size of thegroup and the other will do the sample (part two) in duplicate (or triplicate). This way eachstation will have data for each part. A reference set of data will be provided for comparison.Rotate the work within the group so that different pairs are doing the titrating andrecording of numbers.

Part one: Standard1. Place 10.0 ml (use volumetric pipette) of standard chloride solution in a 250 ml

beaker, dilute to about 100 ml with distilled water, and add 2.0 ml of concentrated

3HNO (use plastic dropper). Nitric acid is in the sink and is very corrosive..2. Immerse stir bar, place beaker on stir plate and lower electrodes into the solution.

Take care that stirrer is not hitting the electrodes.3. Start the stirrer and set the millivolt reading to zero or record initial mv reading

[Note: Some meters can not be set to zero].

34. Begin titrating, in increments, with standard AgNO , recording the volume andmillivolt reading for each increment. At the start, large increments (1-2 mls) may beadded; then, as the end-point of the reaction is approached (ª mV/ml increases),smaller increments (0.1 to 0.2 ml or drop-wise) should be added, until past the end-point when larger increments can be used again (ª mV/ml decreases). Continue thetitration about 5 ml past the end-point. The end-point occurs at the greatest mV

3change per unit addition of AgNO .5. Plot a differential titration curve to determine the exact end-point..

36. Calculate the Normality of the AgNO using the following equation:


Part Two: Sample

1. Measure 100 ml of sample or portion made up to 100 ml, if dilution is necessary intoa 250 ml beaker (use a graduated cylinder to measure).

32. Add 2 ml of HNO and proceed as described in Part One. (If pH of the sample werequite high it would be necessary to first adjust the pH to about 4 and then add 2 ml

3HNO . It is not, in this case.)3. Plot three curves for this titration:

a. Millivolt reading versus ml of titrant.b. Millivolts per ml versus ml of titrant.c. Millivolts per ml per ml versus ml of titrant.

In plotting b and c, which are merely first and second derivatives, be careful to usethe average value of ml of titrant on the abscissa.

4. Calculate the chloride concentration in the sample using the following equation:

3Where: A = ml AgNOB = ml Blank

3N = normality of titrant (AgNO )D = ml of sample

ml mg/L Cl-

From graph a S))))))))Q S))))))))Qb S))))))))Q S))))))))Q c S))))))))Q S))))))))Q


Chloride - Potentiometric Titration

RAW DATA FIRST DERIVATIVE SECOND DERIVATIVE

A

Volume

3AgNO

Added

(Vol) (ml)

B

Meter

Reading (E)

(mV)

C

ª(Vol)

(ml)

D

ª(E)

(mV)

E

ave

3(AgNO )

Volume

(ml)

F

ª(E)/ª(Vol)

(mV/ml)

G

ª(ªE/ªVol)

(mV/ml)

H

ª[Ave(Vol)]

(ml)

I

Ave of

Ave(Vol)

(ml)

J

ª(ªE/ªVol)

/ml

(mV/ml/ml

)

Extra pages are at the back of this lab manual in appendix.


INTERFERENCES IN CHLORIDE DETERMINATIONSPOTENTIOMETRIC METHOD# Iodide, fluoride and bromide titrate as chloride.# Other interferences: ferricyanide, chromate, dichromate, ferric ion.# Pretreatment of environmental samples usually required.MERCURIC NITRATE TITRATION METHOD# Iodide, fluoride and bromide titrate as chloride.# Chromate, ferric, and sulfite ions interfere when concentration greater than 10

mg/L.# Colour may obscure or interfere with end-point determination.# Sample pH may need adjustment beyond the capacity of indicator-acidifier reagent.# Environmental samples usually require complex pre-treatment.COLOURIMETRIC METHOD# Bromide, iodide, fluoride, cyanide, thiosulphate, hydrazine, and nitrite interfere.# Colour in the sample may interfere in the absorbance measurement.# Environmental samples usually require pretreatment.SPECIFIC ION ELECTRODE# All halides interfere (the electrode is a halide electrode).# High levels of ions which form very insoluble salts of silver may deposit a layer of

salt on the electrode membrane, causing electrode malfunction. Also, stronglyreducing solutions may form a surface layer of silver.

# Mercury must be absent from samples.# In practice, specific ion electrodes are rarely usable in environmental samples.KNOWN (STANDARD) ADDITION TECHNIQUE# The known addition techniques will only correct for certain types of interferences.

In the above tests, known addition cannot correct for any ions that test as chlorideion. It can only correct for substances that enhance or suppress the test responseproportional to the amount of chloride ion present.

PRETREATMENT TECHNIQUES# There are no "standard" pretreatment techniques. Each sample type requires

development of an appropriate method which, in itself, requires knowledge of thecompounds in the sample which are likely to interfere. This is a complex, time-consuming process requiring a sound knowledge of chemistry and extensiveexperience working with environmental samples.

Lab 3 Questions

1. In the Mohr method for chlorides (described in Sawyer and McCarty) what wouldthe equilibrium concentration of silver ions be (in mg/L) when the chlorideconcentration has been reduced to 0.3 mg/L?

2. Outline the advantages and disadvantages of all the methods used in this lab tomeasure chloride concentration.

3. From your chloride results, can you see any advantage in suing the first or secondderivative curve? Discuss.


W A R N I N G

Glacial acetic acid is very corrosive. Bleach/chlorine solutions are strong oxidants.Personal protective equipment required at all times when working in the lab are: eyeprotection, gloves and lab coats. When weighing chemicals at the balance, clean up all

spills with a brush into a container and wash crystals down the drain. Wipe up all liquidspills immediately.

Lab o ra to ry 5: Ch lo rin e an d Ch lo rin e De m an d

OBJECT: -to acquaint you with disinfection of water supplies and to demonstratetechniques for the determination of chlorine residuals; to illustrate the conceptsof free and combined chlorine residuals, chlorine demand and break-pointchlorination.

Materials: -600 ml beakers, 250 ml Erlenmeyer flasks, 250 ml volumetric flasks, pipettes,burettes and stands, 100 ml graduated cylinder, bleach or hypochloritesolution, 0.025 N sodium thiosulphate, chlorine free water, (concentrated)glacial acetic acid, potassium iodide (KI) crystals, starch indicator, phosphatebuffer solution, N,N-diethyl-p-phenylenediamine (DPD) indicator solution,standard ferrous ammonium sulphate (FAS) solution.

REFERENCES:Standard Methods 19th ed. 1995. pages 4-56 to 4-57 or older/newer editions.

I. PREPARATION OF STOCK CHLORINE SOLUTION(Iodometric method - total chlorine residual)

1. Prepare a stock solution of strong chlorine water by adding 5 mls of bleach solution (sodiumhypochlorite) to about 200 mls of water in a 250 ml volumetric flask. Make up to the 250 mland invert flask several times to mix. Rinse a burette and fill it with this stock chlorinesolution..

2. Take a 250 ml Erlenmeyer flask and add about 1 gram of potassium iodide, about 50 ml ofchlorine-free water (distilled water will do), about 5 ml of glacial acetic acid (use a graduatedcylinder), and exactly 10 ml of 0.025 N sodium thiosulphate solution (use a volumetricpipette).

3. Mix well (on stir plate with stir bar), add 1 ml of starch solution, allow to mix again. Titraterapidly with the stock chlorine solution (in burette) until the appearance of a constant


purplish-blue colour. 4. From the amount of chlorine solution used, calculate the chlorine concentration in the stock

solution (See Data and Results section for formula).Work in groups of three or four for this part, as there is lots to do - COORDINATE!

IIa.Breakpoint Chlorination using Free and Combined Chlorine Residuals Values1. 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). 2. 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 orderto allow enough time for titrating the free and combined chlorine residuals, refilling burette,etc. Sequentially add the necessary volumes of chlorine stock solution to each beaker at theappropriate time spacing to provide the applied dosages given by the instructor (see board).

3. After 15 minutes of contact time, and again after 1 hour of contact time determine the freeand combined chlorine (see below) in 100 ml samples taken from each of the beakers at theappropriate time spacing (15 minutes and 60 minutes after the addition of the chlorine doseto each beaker).

IIb. Determination of Free and Combined Chlorine by the DPD Method1. Place 5 ml of phosphate buffer solution and 5 ml of DPD solution in a 250 ml conical

(Erlenmeyer) flask and mix. These 6 flasks can be prepared at once to make them ready forthe next step.

2. Add 100 ml of sample or diluted* sample using a 100 ml graduated cylinder and mix again.(*NOTE: If total chlorine exceeds 5 mg/L use a smaller sample and dilute to a total volumeof 100 mls with chlorine-free water. Mix usual volumes of buffer reagent and DPD indicatorsolution with distilled water b e f o re adding sufficient sample to bring total volume to 100 mlor the test will not work. It is likely that the highest dose will require dilution.).a) Free chlorine: titrate rapidly with standard FAS (Ferrous ammonium sulfate) titrant until

the red colour is discharged. Record the burette reading (mls) = Reading A.b) Monochloramine: Add two drops of KI solution (to the same sample) and mix. Continue

titrating until red colour (if any) is discharged once again (Reading B). Go to c.c) Dichloramine: Add about 1 g KI (to the same sample) and mix to dissolve. Let stir for 2

min and then continue titrating until red colour (if any) is discharged (Reading C). Fordichloramine concentrations greater than 1 mg/L, let stand 2 min more if colour drifts backindicating incomplete reaction.

d) Alternatively: (Not to be done in this lab.) Free and combined chlorine or totalchlorine: To obtain total chlorine in one reading, add full amount of KI at the start (ie 2 dropsKI solution and 1 g of KI crystals) to a fresh sample, mix to dissolve, let stand for two minutesand titrate until the red colour (if present) is discharged. Let stand 2 minutes more; if colourdrifts back (indicating an incomplete reaction), titrate to colourless again. Record the burettereading (Reading C).Total chlorine (Free plus Combined) = Reading CFree residual chlorine = Reading ACombined residual chlorine (dichloramine plus monochloramine) = Reading C - A Monochloramine= B-A Dichloramine= C-B

For interferences, please refer to Standard Methods, 18th edition, Method 4500-Cl F. pp 4-43.


DATA AND RESULTS

I. Standardization of chlorine stock solution:Volume of sodium thiosulphate used = ______ mls (A)Normality of sodium thiosulphate used = ______ (N)Volume of stock chlorine solution = ______ mls (B)

2Cl concentration of stock solution = = _________mg/L Cl as Cl *

*To determine bleach concentration as percent you must multiply first by your dilution (i.e. 250)then convert mg/L to g/100 mls = %. Guaranteed analysis is normally printed on the containerand is usually 5-6%.

2II. FAS titrant concentration is 1 ml = 100 µg Cl as Cla. Determine the concentration of total, free and combined residual chlorine for each

applied chlorine dose.b. Make a plot of the three residual chlorine components against the applied chlorine

dosage. Make a separate graph for the 15 minute and 1 hour contact time.c. Discuss your results as a function of the residual forms of chlorine present at different

chlorine dosages and at different contact times. d. 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. Why is it important to determine chlorine residuals in domestic water supplies?

2. Given: HOCl W H + OCl+ -

eqvK = 2.7 x 10 at 25 C-8 o

% HOCl = 27

What is the pH of the solution?

3. Under what conditions is the practice of break point chlorination necessary?

4. a. The rate of kill of bacteria by chlorination follows first-order reaction kinetics. If this istrue, what percentage of bacteria would be killed in 10 minutes at a chlorine residual of0.5 mg/L if 50% are killed in 1½ minutes at this concentration?

b. If a sewage sample contains 10 x 10 coliforms/100 ml, how many coliforms would6

remain after a chlorine contact time of 10 minutes? 20 minutes?


15 MINUTE CONTACT TIME

BEAKER NUMBER 1 2 3 4 5 6

2Vol. of Cl Soln. (mls) (Stock=_____mg/L Cl as Cl )

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


60 MINUTE CONTACT TIME

BEAKER NUMBER 1 2 3 4 5 6

2Vol. of Cl Soln. (mls) (Stock=_____mg/L Cl as Cl )

Applied Cl Dosage (mg/L)

Vol. of FAS to endpoint A (mls)

Vol. of FAS to endpoint B (mls) (includes A)

Vol. of FAS to endpoint C (mls) (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


W A R N I N G

Please use caution when handling all chemicals, particularly those labelled withspecific hazards. Use care when using lab equipment. Probes, metres andcalibrated glassware are available in limited quantities and are very expensive toreplace. Always wear personal protective equipment.

Lab o rato ry 6: Alkalin ity , Ac id ity , p H, Hard n e s s , Co lo u r,Tu rb id ity

OBJECT:

-to familiarize you with the most common water treatment tests; to acquaint you with theuse of pH metres, pH test paper and other water quality assessment equipment.

MATERIALS:

-pH meter, pH test paper, magnetic stirrer, bromphenol blue, phenolphthalein, bromcresolgreen, metacresol purple, standard acid, standard base, burettes and stand, graduated cylinders,beakers, flasks, colour comparator, turbidimeters, EDTA, buffer solution, hardness indicators,1 N NaOH, water samples.

I. DETERMINATION OF pH1. 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. (Take care not tocontaminate paper in case.)

2. Calibrate the pH meter by following the procedure outlined on the instruction sheetbeside the meter unless told otherwise. Be sure that buffers are being stirred when youperform the calibration and that when you change buffers you thoroughly rinse theprobe into a waste beaker and blot dry so that you do not contaminate other buffers oryour samples. Normally, the two buffers that are chosen for calibration will bracket yoursample ie if sample is less than 7, then use buffers 4 and 7 to calibrate; if above 7, thenuse buffers 7 and 10.

3. After calibration is complete, rinse & dry probe, place a beaker containing the sampleand stir-bar on the stir-plate, lower probe, turn on stirrer and obtain a stable reading.(Donot allow the stir bar to hit the probe.) Record value in following table.

4. Rinse probe when you are finished and leave immersed in pH 7 buffer for storage.


II. DETERMINATION OF ALKALINITY

1. Rinse and fill a labelled burette with standard acid solution. 2. Measure 100 ml of water sample into a clean, sample-rinsed graduated cylinder and

transfer to an Erlenmeyer flask. In order to remove any free residual chlorine (whichinterferes with the indicator colour response), add 1 drop of 0.1 N sodium thiosulphateto the sample. Add a stir-bar.

3. Add a full dropper of phenolphthalein (P) indicator and if the solution stays pink orred, titrate until just colourless. Record burette reading (P). (If solution is clear afteraddition of P...think what that means!)

4. Add a dropper full of bromcresol green (BG= MO) to this same water sample andcontinue the titration until the colour changes from blue to yellow (at pH 4.5).

5. Record the burette reading in the following table. (BG or MO)6. If sample is highly alkaline (titration goes over one burette full) dilute the sample and

titrate again. Apply dilution factor to obtain final result.

III. DETERMINATION OF ACIDITY

1. Rinse and fill another burette with standard base solution and label it.2. Measure 100 ml of sample (with minimum agitation or exposure to air) using a graduated

cylinder and transfer carefully to an Erlenmeyer flask. Add 1 drop of sodiumthiosulphate solution.

3. Add a full dropper of bromphenol blue indicator and titrate until the colour changesfrom yellow to blue ("MO" Acidity). (If the solution is already blue after indicator....?)

4. Measure out a new sample and add 1 drop of sodium thiosulphate and a full dropper ofphenolphthalein. Titrate to the first uniform pink colour (colour stays). Record thevolume of titrant required in the following table.

IV. TOTAL HARDNESS DETERMINATION - EDTA TITRIMETRIC METHOD

1. Rinse and fill a burette with standard EDTA solution and record the initial burettereading.

2. Measure out 25 mls of sample using a graduated cylinder and dilute to 50 ml withdistilled water. Pour into a 250 ml Erlenmeyer flask. Add stir-bar.

3. Add 1-2 ml of buffer solution using dropper bottle (3 full droppers) and one aliquot ofTotal Hardness Indicator using the special dispenser on the chemical bottle. The buffer

3elevates the pH to 10. A pH much above 10 promotes CaCO precipitate. 4. Titrate slowly with EDTA, while stirring, until the reddish tinge disappears from the

solution and a pure blue remains. Add the last few drops at 3-5 second intervals so thatyou do not "overshoot" the end-point. White paper placed under the flask will facilitatethis determination. Complete the titration within 5 minutes to minimize the

3tendency toward CaCO precipitation. If a ppt does form within the 5 minutes, itmay be necessary to repeat the titration with the sample diluted 1 to 1 (again) withdistilled water. Alternatively, if the approximate hardness is known (by unsuccessfulattempts), add 90% of the titrant to the sample before adjusting the pH with buffer.


5. Record the final burette reading in the following table.

V. CALCIUM HARDNESS DETERMINATION - EDTA TITRIMETRIC METHOD

1. Refill the burette with EDTA titrant and note the initial reading.2. Measure out 50 ml of sample into an Erlenmeyer flask and add 2.0 ml of 1 N NaOH

solution (use 3 droppers full), or a volume sufficient to produce a pH of 13-14 (designedto precipitate the magnesium as a hydroxide). Add a stir-bar and stir to mix.

3. Add one aliquot of the Calcium Hardness Indicator and titrate with EDTA slowly, withcontinuous stirring to a pure blue end-point.

4. Record the final burette reading in the following table.

VI. COLOUR - TRUE AND APPARENT

PART A: TRUE COLOUR - HACH Spectrophotometer. - Enter the stored program numberfor true colour=1201. Filter about 20 mls of sample using a filter funnel and stand and the pre-fluted filter paper

provided2. Pour the supernatant into one of the cells (to the etched mark) and fill another with distilled

water.3. Place the cell containing water into the spectrophotometer and press Zero. 4. Place the cell containing sample in it and press Read. The instrument may suggest diluting if

over range.. Values are reported as APHA Colour Units (eqv. to mg/L platinum aschloroplatinate)

PART B: APPARENT COLOUR1. Use the technique outlined in Part A, except on an unfiltered sample, to determine the

apparent colour (measures the influence of turbidity on the reading). The reading might behigher so if you had to dilute in Part A dilute for Part B.

2. Record all colour data in the following table.

VII. TURBIDITY - BY HACH 2100P Turbidity Meter. Demonstration of JacksonCandle and HelligePART A: Hach Turbidimeter1. Fill a sample cell to the line (about 15 mL), taking care to handle the sample cell by the top.

Cap the cell..2. Wipe the cell with a soft, lint-free cloth to remove water spots and fingerprints.3. Press: I/O. The instrument will turn on but will turn off automatically after a short time

so you should be ready.4. Insert the sample cell in the instrument cell compartment so the diamond or orientation

mark aligns with the raised orientation mark in front of the cell compartment. Close the lid.Select automatic range by pressing the RANGE key. The display will show AUTO RNG when the instrument is in automatic range..

5. Select signal averaging mode by pressing the SIGNAL AVERAGE key. The display willshow SIG AVG when the instrument is using signal averaging. Use signal average mode if


the sample causes a noisy signal (display changes constantly).6. Press: READ The display will show - - - - NTU, then the turbidity in NTU. Record the

turbidity after the lamp symbol turns off..

PART B: Hellige Turbidimeter - Demonstration only1. Carefully fill the 20 mm viewing depth tube to the mark with sample. Lower the plunger

into the liquid making sure no bubbles are trapped under the plunger and the top is dry.Wipe the outside of the tube to dry it and place it in the turbidimeter (in the circulargroove of the mirror unit). Note that the rectangular door mirror is closed, that the filterselector shows "none" and that bulb B is in use. Close the door and switch on the light.

2. Immediately balance the light intensity of the central spot with the surroundingobservation field by turning the dial on the right-hand side of the instrument. Take thereading where the dark central part first merges with the surrounding field. If you taketoo long, the turbidity may start to settle and condense on the bottom of the tube.

3. Read the scale on the dial, record the number and refer to the appropriate graph to

2determine the turbidity as mg/L SiO .

PART C: Jackson Candle Turbidity - demonstration only..

1. Be sure that the calibrated tubes are clean before adding samples to the tubes. DO NOT PLACE AN

EMPTY TUBE IN THE TUBE HOLDER WITH THE CANDLE LIT - adding liquid to a hot

tube will cause it to shatter. Try to be quick and not burn the candles more than a few minutes at a

time. Pinch all excess charred wick with fingers before lighting or soot may be deposited on the glass

tubes, obscuring vision.

2. Pour sample into the tube in small increments until you can no longer distinguish the shape of the candle

flame. 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. (This allows you to make a more gradual and accurate

approach to the end-point.)

3. Remove the glass tube from the holder and read the turbidity at the meniscus of the sample. Note that

there are several different scales on the side of the tube. Record value in following table as J.T.U.s.

Result of Titration

Hydroxide Alkalinity

3as CaCO

Carbonate Alkalinity

3as CaCO

BicarbonateConcentration

3as CaCO

P=0P<1/2TP=1/2TP>1/2T

P=T

000

2P-TT

02P2P2(T-P)0

TT-2P

000

Where: P = Phenolphthalein alkalinity; T = Total alkalinity.See Standard Methods, 16th edition, page 272-273.


DATA

Sample 1 Sample 2

pH - Initial pH by test paper __________ ___________

Initial pH by pH meter __________ ___________

ALKALINITY- Sample volume __________

P. Alkalinity end-point reading (mls) __________

"M.O." Alkalinity end-point (mls) __________

Initial burette reading __________

Net P. Alkalinity titre (mls) __________

Net "M.O." Alkalinity titre (mls) __________

ACIDITY- Sample volume __________ ___________

"M.O." Acidity end-point reading (mls)__________ ___________

Initial burette reading __________ ___________

P. Acidity end-point reading (mls) __________ ___________

Initial burette reading __________ ___________

Net "M.O." titre (mls) __________ ___________

Net Total acidity titre (mls) __________ ___________

2Net CO Acidity titre (mls) __________ ___________

TOTAL Hardness Sample volume (mls)__________

Final burette reading (mls) __________

Initial burette reading (mls) __________

Net titre (mls) __________

3Total Hardness as mg/L CaCO __________

CALCIUM Sample volume (mls) __________

Final burette reading (mls) __________

Initial burette reading (mls) __________

Net titre (mls) __________

Calcium as mg Ca /L __________++

3Calcium hardness as mg CaCO /L __________


CALCULATIONS

Calcium:

Calcium Hardness:

where: A = ml titrant for sample

3B = mg CaCO equivalent to 1.00 ml EDTA titrant at the calcium indicator

endpoint = 1.0

Total Hardness (EDTA):

where: A = ml titrant for sample

3B = mg CaCO equivalent to 1.00 ml EDTA titrant at the calcium indicator

endpoint = 1.0

Alkalinity:

where: A = ml standard acid used

N = normality of standard acid

Acidity:

where:A = ml standard base used

N = normality of standard base


RESULTS (sample)

3P. Alkalinity as CaCO (mg/L) ______________________________________

3"M.O." Alkalinity as CaCO (mg/L) ______________________________________

3OH Alkalinity as CaCO (mg/L) ______________________________________-

3 3CO Alkalinity as CaCO (mg/L) ______________________________________-2

3 3HCO Alkalinity as CaCO (mg/L) ______________________________________-

OH Alkalinity as OH ion (mg/L) ______________________________________- -

3 3CO Alkalinity as CO ion (mg/L) ______________________________________-2 -2

3 3HCO Alkalinity as HCO ion (mg/L) ______________________________________- -

3"M.O." Acidity as CaCO (mg/L) ______________________________________

3Total Acidity as CaCO (mg/L) ______________________________________

2Free carbon dioxide as CO (mg/L) ______________________________________

2Total Acidity as CO (mg/L) ______________________________________

Ca Hardness as mg Ca (mg/L) ______________________________________+2

3Ca Hardness as CaCO (mg/L) ______________________________________

3Mg Hardness as CaCO (mg/L) ______________________________________

Mg Hardness as Mg (mg/L) ______________________________________+2

3Carbonate Hardness as CaCO (mg/L) ______________________________________

3Non-carbonate Hardness as CaCO (mg/L) ______________________________________

3Excess alkalinity as CaCO (mg/L) ______________________________________

Colour - APHA Colour Units: True ______________________________________

Apparent ______________________________________

Turbidity - Hach - Units= N.T.U. ______________________________________


Lab 6 Questions

1. Is this water suitable for a domestic water supply? Why?

2. What errors can occur in the calcium hardness determination? Why?

3. What sanitary significance does colour have?

4. A water has the following analysis:

3Na 15 mg/L HCO 70 mg/L Mg 18 mg/L+ - +2

K 33 mg/L Cl 42 mg/L+ -

4Ca 12 mg/L SO 8 mg/L+2 -2

3What is the carbonate, non-carbonate and total hardness of this water in mg/L as CaCO ?in meq/L? Is the analysis complete? Why?

6. From equations (17-12) and (17-13) in Sawyer and McCarty, calculate the carbonate

3 2alkalinity in mg/L as CaCO for the water sample analyzed during this lab, given K = 4.7

wx 10 and K = 10 .-11 -14

Does this agree with the results from Part II of this lab? Explain.

Note: You should bring a copy of this lab to the next two sessions as you will needthe same instructions to perform the same tests in the Coagulation and SofteningLabs.


ATTENTION

This lab will require patience and cooperation from all of you as we have onlytwo jar testers with 6 paddle sites each. Please try to coordinate your work withthe rest of the class. You should have with you the part of Exercise 7 that givesinstructions for the various tests.

Lab o rato ry 7a: Co ag u latio n and 7b : Co ag u latio n &So fte n in g

OBJECT:-to illustrate the principles of coagulation and water softening. This is a two-step

experiment, to be conducted over two lab periods. The first step will be to analyse theraw water sample and the supernate from a number of alum dosages (to be announced inthe class) using the techniques learned in Lab. No.6. The optimum alum dosage forcoagulation of the sample will then be chosen.for the next session (b). Several lime andsoda ash dosages will also be selected for determining the efficiency of water softening.In the second lab you will use the previously determined dosages to coagulate and softenthe sample and then re-analyse the resultant water.

MATERIALS:

2 4 3 2-Jar Test Apparatus, 1000 ml beakers, alum solution (Al (SO ) @18H O), soda ash

2 3 2solution (Na CO ) and lime (Ca(OH) ) or sodium hydroxide (NaOH), and all of thematerials from Lab. No. 7 (except Jackson Candle and Hellige Turbidimeter).


Part A:. COAGULATION USING ALUM - work in 2 or 3 teams to assess a total of 6dosages for each team.

1. Measure out six 1000 ml aliquots of sample (using graduated cylinder to measure,not a beaker ) and transfer to 1000 ml beakers.

2. Place beakers on the jar tester apparatus. You may need to use blocks to positionthe paddle 0.5 to 1 cm off the bottom of the beaker. Make sure it is centred.

3. Add the assigned alum dosages as quickly as possible and then start the stirrer. Stirat 100 rpm for 1 minute, 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 issheared). Observe and make notes describing floc formation and note the time ofappearance.

4. Stop stirring. Lift paddles out of beakers and secure. Record relative floc sizes(pin-point, small, medium, large); clarity of supernatant liquid (very clear, clear,hazy, cloudy); and settling characteristics of the floc (rapid, moderate, slow).

5. After the flocs have settled (allow 15 min), decant the supernatant liquid carefullyinto another set of beakers (try not to stir up what has settled).

6. Analyze these supernates plus a sample of raw water for pH, alkalinity, acidity,hardness (total and calcium), true colour and turbidity (Hach). A pH meter couldbe used for the alkalinity and acidity determinations, if available, but because of equipment limitations, use indicators as specified in Lab 6.

7. Complete the coagulation summary sheet during the lab period, if time permits,and fill in the table on the board. In the lecture, you will discuss the data andselect the appropriate alum dosage to be used by all groups in the water softeningstep next week. You will also determine the optimum lime and soda ash dosagesto soften this water and select a range of dosages surrounding this optimum forinvestigation next week.

Part B: WATER SOFTENING PROCEDURE (to be done the following week) -again work as teams of 2 or 3.

1. Pour six 1000 ml aliquots of sample into 1000 ml beakers and place on the jar teststirrer apparatus. Add the appropriate alum dosage and immediately start stirring.Stir at 100 rpm for 1 minute. Reduce the stirring rate to about 20 rpm for 10minutes. Stop stirrer and allow floc to settle.

2. Carefully decant into a graduated cylinder 800 mls of the supernatant liquid andpour into fresh beakers, position them under stirrers. Discard the floc and cleanbeakers in preparation for a second decanting in Step 4. Add the designated lime(or NaOH) and soda ash additions as quickly as possible to the decantedsamples. (NB dosage now based on 800 ml sample size). Stir at 100 rpm for 1minute and at 20 rpm for 10 minutes. Make relative estimates of floc sizes, timesof appearance and descriptions and record.

3. Stop stirrer. Record observations on settling rate, floc volume, and clarity ofsupernatant liquid.

4. After the floc has settled (allow 15 min), decant the supernatant liquid carefullyinto a clean set of beaker (try not to stir up what has settled) and discard the floc..

5. Analyze the supernates for pH, alkalinity, acidity, hardness (total and calcium),turbidity and colour.

6. Determine which dosages were most appropriate for this sample.

Brief Methods Summary for Analyses

Alkalinity:@ 100 ml of sample, drop of 0.1 N sodium thiosulphate


@ titrate with 0.02 N acid with phenolphthalein to colourless - pH 8.3 and withbromcresol green (continuing with the same sample) to yellow - pH 4.5.

3@ calculation - alkalinity (mg/L CaCO )= mls titre x N acid x 50,000/mls sample

Acidity:@ 100 ml of sample, drop of 0.1 N sodium thiosulphate@ titrate with 0.02 N base with bromphenol blue to blue - pH 3.7 ("methyl orange"acidity) and with phenolphthalein (using a fresh sample) to pink - pH 8.3("phenolphthalein" or total acidity)

3@ calculation - acidity (mg/L CaCO )= mls titre x N base x 50,000/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 - pure blue).

3@ calculation: hardness (mg/L CaCO )= 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 NaphtholBlue)@ titrate quickly with EDTA to blue end-point (no pink/purple tinge - pure blue).

3@ calculation: Ca hardness (mg/L CaCO )= mls titre x 1000/mls sample

Lab 7 Questions

A. Coagulation1. Analyze the response of colour and turbidity removal as a function of alum dose.2. 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 compensatefor the alkalinity consumption by 70 mg/L alum?

B. Softening1. Based on your chemical analysis of the raw water, show by calculation the theoretical

amount of lime and soda ash required for excess lime/soda ash softening.2. Compare your experimental softening results to the theoretical results based on

solubility equations or mass balance equations.3. Compare removal of hardness components under the different doses - use graphical

presentation.4. What treatment would you recommend to prepare this water for human consumption?

Why?

50September 2007 CIVL 407 Lab M anual

Coagulation Raw Data Sheet

DATA Raw

Sample

Alum

mg/Lmg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L

pH

Alkalinity-sample vol ml

Net titre to pH 8.5 “P”

Net titre to pH 4.5 “TOT”

Acidity - sample vol ml

Net titre to pH 3.7 “MO”

Net titre to pH 8.5 “TOT”

Total Hardness sample vol

Net titre

Ca Hardness - sample vol

Net titre

Notes:


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

3Alkalinity (mg/L CaCO ) “P”

3Alkalinity (mg/L CaCO ) “TOT”

3Acidity (mg/L CaCO ) “MO”

3Acidity (mg/L CaCO ) “TOT”

3Total Hardness (mg/L CaCO )

3Ca Hardness (mg/L CaCO )

Apparent Colour (A.P.H.A.)

True Colour (A.P.H.A.) (filtered)

Turbidity (N.T.U.) unfiltered

Notes:


Coagulation and Softening Raw Data

DATA Lime/NaOH mg/L

Soda Ash mg/L

Alum mg/L

pH

Alkalinity-sample vol ml

Net titre to pH 8.5

Net (Total) titre pH 4.5

Acidity - sample vol ml

Net titre to pH 3.7

Net (Total) titre pH 8.5

Total Hardness sample vol

Net titre

Ca Hardness - sample vol

Net titre

Notes:


Coagulation and Softening Summary Sheet

Data Lime/NaOH mg/L

Soda Ash mg/L

Alum mg/L

pH

3P- Alkalinity -mg/L CaCO

3*Total Alkalinity-mg/L CaCO

3“MO” Acidity -mg/L CaCO

3*Total Acidity -mg/L CaCO

3Total Hardness -mg/L CaCO

3Ca Hardness -mg/L CaCO

3Mg Hardness -mg/L CaCO

3**Carbonate Hard. -mg/L CaCO

3**Non-Carb Hard -mg/L CaCO

3**Excess Alk. -mg/L CaCO

Apparent Colour (A.P.H.A.)

True Colour (A.P.H.A.)

(Filtered)

Turbidity (N.T.U.)

Note:* Total Alkalinity must include Phenolphthalein Alkalinity just as Total Acidity must include “MO” Acidity. ** ByCalculation


4Appendices see: CIVL407Manual_CourseNotes.pdf1. Instructions for Laboratory Report Writing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 552. Reading and Reference Material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 573. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 604. Normal Solutions and Equivalent Weights . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 645. Standard Solutions, Titrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 676. Typical Problems for Acid-Base and General Chemistry . . . . . . . . . . . . . . . . . . . . . 687. Residues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 698. Solids Removal in Wastewater Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 709. DO, BOD, COD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7110. Balancing an Oxidation Reduction Equation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7211. Hach Absorption Method - COD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7312. Chloride Analytical Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7413. Known Addition Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8014. Bacteriology and Water-related diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8215. Faecal Coliforms - Levels and Water Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8316. Bacteriological Examination - Additional Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . 8417. Chlorine - Chlorine Demand . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9118. Equilibrium Relationships of Chlorine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9219. Acidity, Alkalinity, pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9420. Concepts and Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9621. Alkalinity Species as a Function of pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10022. Colour, Turbidity, Hardness, Coagulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10223. Hardness Complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104

324. Milliequivalent and mg/l as CaCO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10525. Water Softening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10726. Chemical Requirements for Water Softening . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11027. Bar Diagram Method for Water Softening Problems . . . . . . . . . . . . . . . . . . . . . . 11228. Periodic table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11729. Extra tables for Chlorine Lab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118

CIVL407LABMANUAL_20072

Documents