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experiment13LECTURES AND LAB SKILLS EMPHASIZED

• Application of analytical techniques to a real-world problem

• Sampling technique

• Preparation of standards

• Multiple trials

• Stoichiometry

• Titration

• Beer’s law

Water Quality Analysis

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IN THE LAB

• Students will work in pairs.

• Parts must be completed in order.

• Record your procedure and original data in your lab notebook along with your calculations.

• Report data collected and subsequent calculations to www.chem21labs.com.

• All equipment should be returned to the correct location after use.

WASTE

• Dispose of the NitraVer solution residue ONLY in the appropriately labeled container.

SAFETY

• Gloves and safety goggles are mandatory when anyone is performing an experiment in the lab.

• Water collection: When collecting water samples, use common sense about the sampling site.

• Do not trespass on private property.

• Do not collect samples from rapidly running streams unless you can approach them safely.

• Do not collect samples unsafely.

• Do not damage plants and animal habitat on the stream banks.

• Watch for poison ivy, snakes, and other potential hazards.

• Always take away all materials you bring to the sampling site back with you.

• The Water Hardness Buffer contains ammonia. Please be aware of any sensitiveness you may have.

• Take care in handling solid reagents to avoid inhalation and eye exposure. Always use a brush to clean the balance.

• Wear long pants, closed-toed shoes, and shirts with sleeves. Clothing is expected to reduce the ex-posure of bare skin to potential chemical splashes.

• Always wash your hands before leaving the laboratory.

• Dispose of the ammonia-containing solutions in the container marked for “water hardness.”

• Dispose of all other solutions in the aqueous container.

Additional information can be found at: https://wp.as.uky.edu/genchem/home/che-113/13-titration/

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starts with the container you select to collect your sample in. Plastic is best for this case, as it is most likely to not react with any chemical species in the sample. Your container should also be thoroughly cleaned and dried before you add any of your sam-ple to it. When collecting your sample, try not to disturb any of the surrounding environment and leave your collection site in better shape than how you found it. Make sure when you dip your col-lection container into the water that you do not disturb the sediment at the bottom of the water source and avoid collecting algae in your sample collection container. Replace the lid and label your container with its contents, the location it was col-lected, date, and time.

Often more than one sample is needed to ensure that the data collected is reflective of the environ-ment as a whole. Samples could be collected at multiple water depths or different distances from shore. Some studies may also require samples to be collected from different locations amongst the source water. Remember, it is important that you try not to disturb the water too much. When collecting your sample, start at the lowest depth you are sampling from, taking care not to disturb sediment, and gradually move toward the surface while filling the bottle.

Rinse the sample bottle a few times with stream water, dumping the water out downstream from the area the sample will be collected. Hold the mouth of the bottle upstream and fill the bottle completely.

Figure 13.1. The Kentucky River watershed.

Watersheds in KentuckyWatersheds consist of the water that is under, on, and drains across an area in order to drain into the same place. They consist of lakes, streams, rivers, ground water, drinking water, stormwater, wetlands, and wastewater sources. Kentucky has 48 watershed areas1 grouped into seven water ba-sins.2 In the Lexington area, the Kentucky River Basin drains Fayette County and is monitored by volunteers who are part of the Kentucky River Watershed Watch. This group regularly goes out into the Kentucky River Watershed to collect water samples to test for water quality. You are going to collect water samples from the Lexington area and complete some of the water tests that this group collects.

Sampling TechniquesThe samples you have worked with so far have been pure samples taken directly from reagent containers in the lab. These samples have gener-ally contained only one substance and very limited contamination from other reactive substances. With this lab, however, you will need to collect a sample from a much more varied source.

When collecting samples, it is extremely impor-tant that care is taken to ensure that you are not introducing contaminants to your sample. This

1 US Environmental Protection Agency. “Surf Your Watershed.” http://cfpub.epa.gov/surf/state.cfm?statepostal=KY (accessed Dec. 7, 2017).2 Kentucky Department of Environmental Protection Division of Water. Division of Water - Basins. http://water.ky.gov/watershed/Pages/default.aspx (accessed Dec. 7, 2017).

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Once a sample is removed from the body of water, its temperature, dissolved oxygen, and pH chang-es. Chemical reactions can continue in the sample, changing the concentrations of other substances as well.

Turbidity and WaterWater seen in streams and sometimes coming out of the tap can appear cloudy in nature. That “cloudiness” can be quantified using a light scat-tering technique known as turbidity. Because the amount of light scattering seen can be dependent on the size of the particles, care must be taken to ensure that the sample is stable (particles are not settling into a solid at the bottom). For our case, that can easily be solved by gently inverting the container before the measurement is made.

To measure turbidity, light is passed through the sample and information about the reflected light collected at a detector. Nephelometry measures scattered light at a detector located 90º from the sample. The turbidimeter we will use will be mea-suring the light emitted from an IR LED scattered at 90º from the sample. Due to the positioning of the LED within the instrument, only the R(Turb.) slot will be used on the probe. The higher the in-tensity of scattered light, the higher the turbidity.

The turbidity values are reported in nephelomet-ric turbidity units (NTU). Low NTU values have less light scattering and are generally less cloudy, while those with higher NTU values have more light scattering and are more cloudy. In order to calibrate the probe, we will be using three turbidity standards. You’ll need to plot the turbidity signal versus the turbidity standard and should have a well-fitting linear curve.

lightsource

sample detectornephelometry

©Hayden-McNeil, LLC

detectorturbidity

Figure 13.2. Function of turbidimeter.

The cloudiness in the water can be due to a num-ber of types of particles, including clay, silt, inor-ganic and organic matter, algae, soluble organic compounds, plankton and other microscopic organisms.3 While the cloudiness is not necessar-ily aesthetically pleasing, it may also be a source of health concern, as the particles may provide a home for pathogens. When the turbidity is not re-moved before human consumption, these patho-gens can grow throughout the water distribution system, leading to waterborne diseases, which can cause significant gastroenteritis.

Additional information on turbidity can be found at https://www.epa.gov/dwreginfo/guidance-manuals- surface-water-treatment-rules or https://www.epa.gov/ground-water-and-drinking-water.

Water HardnessAs water flows through deposits of limestone and other calcium and magnesium containing rock, it dissolves up these minerals. Those minerals over time come out of solution and form the discolor-ation on your shower stall called scale. While we may see the presence of dissolved ions in the water as a detriment, fish use them through absorption for bone formation, blood clotting, and other met-abolic processes.4

As limestone, made predominately of calcium carbonate, dissolves, the calcium ions are released

3 US Geological Survey Turbidity. http://water.usgs.gov/edu/ turbidity.html (accessed May).4 Wurts, W. A., Understanding Water Hardness. World Aquaculture 1993, 24 (1), 18.

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into the water. You will need to titrate your water sample to determine how much calcium ions are dissolved in the water. To do this, you will perform a complexometric titration using ethylenediam-minetetraacetic acid (EDTA) whose chemical for-mula is H4C10H12N2O4. EDTA is a complexing, or chelating, agent that is used to capture metal ions, removing the metal ions.

C

O

O

O

O

O

Ca2+

NN

C

C

C

C

CH

H

H

H

H

H

H

HH

H C

C

H

H

O

C

O

O

C

Ca2+ – Ethylenediaminetetraacetate complex

©Hayden-McNeil, LLC

As EDTA is not very water soluble, we will be us-ing the disodium salt, Na2H2C10H12N2O4. EDTA reacts with Ca2+(aq) in the following way. For sim-plicity, Y will represent C10H12N2O4, thus EDTA will be written as Na2H2Y.

H2Y2–(aq) + Ca2+(aq) → CaY2–(aq) + 2 H+(aq)

You will first need to adjust the pH of the sample to pH 10 using the “water hardness test buffer” solu-tion, a preparation of ammonium chloride in am-monia. This is to help ensure that the EDTA will only react with Ca2+ and Mg2+ ions over any other metal ions, like Fe3+ that may be present in the sample, by forcing EDTA into the H2C10H12N2O4 form.

As we are using a buffered solution, we cannot use the MeasureNet pH probes to monitor pH change, however the calmagite indicator will change color

at the equivalence point due to its tendency to bind less strongly to the metal ion than EDTA. When the indicator is added to the solution, it binds with the metal ion. As we add EDTA, the indicator is displaced from the metal, causing a change in the indicator that is detected due to its change of color. The color change happens very quickly near the equivalence points but is very distinct, in this case going from the red to light purple to a vivid blue when you pass the equivalence point. You will need to titrate slowly, as only a drop of the indica-tor/EDTA solution is necessary to change the col-ors around the end point.

CaIn + EDTA → CaEDTA + In (red) (colorless) (colorless) (blue)

The U.S. Geological Survey classifies water hard-ness based upon the amount of calcium carbonate dissolved in water: 0 to 60 mg/L as soft, 121 to 180 mg/L as hard.5

Phosphates in WaterLiving organisms derive their energy from the molecule adenosine triphosphate (ATP). ATP consists of three phosphates bonded to the nucleic acid adenosine. Energy is derived from breaking the bond between each phosphate group, while energy can be stored by forming new phosphate bonds. You need so much ATP that you burn an amount roughly equivalent to your body weight each day.

O

H

H H

OH OH

H

©Hayden-McNeil, LLC

NN

NH2

NNP O–O

O–

O

P O

O–

O

P O

high energy bonds

O–

O

Figure 13.3. Adenosine triphosphate (ATP).

5 US Geological Survey Water Hardness and Alkalinity. http://water.usgs.gov/owq/hardness-alkalinity.html (accessed July 22).

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Table 13.1. Preparation of standard solutions from 0.001 M phosphate solution.

SampleApproximate

ConcentrationApproximate Amount of Phosphate Solution (mL)

Approximate Amount of Water (mL)

Total Volume (mL)

A 2 × 10–5 M 20

B 5 × 10–5 M 20

C 1 × 10–4 M 20

D 2 × 10–4 M 20

E 5 × 10–4 M 20

Beer’s Law Plot0.4

0.3

0.2

0.1

0.0

0 1 2 3 4 5

Abs

orba

nce

Concentration (ppm)

Figure 13.4. Beer’s law plot.

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Animals can easily obtain the necessary amount of phosphate by eating other living organisms; however, plants must obtain it from the ground and often have difficulty doing so. We artificially add phosphate to gardens, yards, and farms using fertilizer. If too much phosphate is added to the environment, runoff can go into the water system. While the aquatic plants will initially benefit from obtaining phosphate easier, the rapid growth can lead to eutrophication, which is characterized by rapid plant growth, followed by the creation of more dead plants for decomposition, a rise in bac-teria growth and oxygen use in the water, which will lead to less oxygen being available for fish, insects, mussels, and other animals, leading to a massive die-off.

We can determine phosphate concentration in water samples using spectrophotometry, which allows us to measure how light interacts with both known and unknown concentrated solu-tions. Phosphate itself does not absorb light, so it will be reacted with ammonium molybdate to create a blue-colored complex which will absorb light in the visible light region. The intensity of the color will be proportional to the amount of blue complex that forms, which, in turn, indicates the amount of phosphate present in the solution. We can determine the concentration of an unknown concentration solution by comparing the intensity of the unknown concentration with a standard curve, created by measuring the intensity of light absorbed by the known concentration solutions.

The EPA monitors phosphate levels in terms of parts per million or ppm. This is equivalent to mil-ligrams per liter. The methods we describe here are very similar to the ones the EPA suggests for volunteer groups.

Nitrate in WaterFertilizer runoff is also a major source of nitrates in water. Congress set maximum contaminant lev-el goals (MCLG) for many substances as part of the 1974 Safe Drinking Water Act and gave the EPA the power to monitor and report the level of these substances.6 For nitrates, the MCLG is 10 mg/L or

6 US Environmental Protection Agency Basic Information about Nitrate in Drinking Water. http://water.epa.gov/drink/contaminants/basicinformation/nitrate.cfm (accessed June 3).

10 ppm. Drinking water with excess nitrates can cause illness in infants under 6 months and, if left untreated, can lead to death.

To test for nitrates, you will use a colorimeter test kit prepared by Hach which uses the cadmium re-duction method, in which cadmium metal is used to reduce nitrates (NO3

–) to nitrites (NO2–).

NO3– + Cd + 2H+ → NO2

– + Cd2+ + H2O

When the nitrite reacts with the other materials present in the pillow packet provided by Hach, it will produce an amber color that can be compared with the color wheel to determine the concentra-tion (mg/L) of nitrate in the solution. Note what units the color wheel measures in.

Total Dissolved SolidsDissolved solids, such as calcium, chlorides, ni-trate, phosphorus, iron, sulfur, and other ion par-ticles, and suspended solids like silt, clay, plankton, and algae, can have an effect on the water balance in cells of marine life.7 Misbalances in the water intake can cause marine life to not be able to main-tain the appropriate density and begin to float or sink more in the water column. Sometimes to the extent that it moves out of the depth for which it is adapted, which could lead to death.

In addition, dissolved solids can have an effect on the clarity and taste of drinking water. Water clar-ity can affect not only the aesthetics in your glass, but also the ability for plants to obtain energy through photosynthesis. Higher concentrations of total solids can also have an effect on wastewater treatment plant’s ability to treat the water and re-turn it to a stream.

The amount of total dissolved solids is typically measured using electrical conductivity. Pure water has poor conductivity. If ions are dissolved in wa-ter, then the conductivity will increase. The EPA, through the National Secondary Drinking Water Regulations, set non-mandatory water quality standards for total dissolved solids at 500 mg/L,

7 US Environmental Protection Agency 5.8 Total Solids. http://water.epa.gov/type/rsl/monitoring/vms58.cfm (accessed June 4).

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though it does not enforce the secondary regu-lations.8 Hard water specifically measures the amounts of dissolved calcium and magnesium ions in water, so is related to total dissolved solids.

Conductivity is approximately related linearly to the total dissolved solids following

Conductivity = TDS * A

where the TDS is in parts per million and the con-ductivity is in units of μS ___ cm .9

The constant, A, can vary from 0.556 to 0.75, de-pending on the exact ions dissolved in water. For our work, we will use the more common value of 0.67.

Materials and Procedures500 mL plastic sample containerMeasureNetColorimeter probecuvettes with caps Universal pH stripsthermometerscuvettes and capsturbidity standards for 1, 10, and 100 NTUwater hardness buffercalmagite indicator solution0.010 M EDTA0.100 M phosphate stock solutionammonium molybdate solutionstannous chloride100 mL volumetric flasktest tubesHach nitrate test strips0.01 M KCl conductivity standardKimwipes

8 US Environmental Protection Agency Secondary Drinking Water Regulations: Guidance for Nuisance Chemicals. http://water.epa.gov/drink/contaminants/secondarystandards.cfm (accessed June 4).9 Fitts, C. R., Groundwater Science. Academic Press: San Diego, CA, 2002.

ProcedureYou’ve been asked by local residents of Lexington to help complete a study on the water quality of local water sources. To assist with this project, you will need to obtain a water sample from a lo-cal source and describe its source. Water collected from the tap anywhere on campus is not accept-able. This description will need to include the GPS coordinates of the position the sample was taken, a description of the location, including surrounding vegetation and wildlife, time and date the sample was taken, the current temperature of the water, and the pH of the water.

You will need to write your data, observations, pro-cedure, and calculations in your lab notebook. All of your data will need to be entered onto Chem21 so it can be used as part of a larger study describing the water quality around Lexington and the sur-rounding communities.

Once you have completed the following tests, you will need to produce a report describing the results of your experiment to the residents in the surrounding community who interact with the watershed. Just like with your lab report, you will need to include an explanation of the chemistry involved with the test, the results of the test, and what it means to the residents. You will also need to show whether or not your data is reproducible. You should also include a list of “credits” that con-tain any citation of material that you used that was not your own.

On-Site

Label Your ContainerAll samples need to be labeled with your name, section number, TA name, date collected, what it is (“water sample from ____”) and “NOT FOR HUMAN CONSUMPTION.” Any markings that are in conflict with this information need to be removed (i.e., the label on a water bottle needs to be removed). Bring this container with you to lab, keeping in mind that any container that you bring into lab is now contaminated and must be disposed of in the lab garbage cans.

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pHDetermine the pH of the water at your sample source.

TemperatureDetermine the temperature of the water source at the time of your sampling.

In-Lab

Water HardnessWater hardness will be determined through titra-tion. To prepare your water sample, you will need to add 2.0 mL of Water Hardness Buffer, approxi-mately 10 drops of calmagite indicator solution, and 2–3 ascorbic acid crystals to a 25 mL aliquot of your water sample. Make sure you record all ob-servations of your water sample as you titrate the solution with EDTA. You will need 3 successful titrations for data analysis.

Phosphate DeterminationPrepare 100 mL of a 1.00 × 10–3 M phosphate so-lution using the 0.1 M stock solution.

Create a standard curve, using 5 solutions of known concentration:

2.00 × 10–5 M, 5.00 × 10–5 M, 1.00 × 10–4 M, 2.00 × 10–4 M, and 5.00 × 10–4 M

You will need 20 mL of each solution.

To form the colored phosphate complex, you will need to add 1.00 mL of ammonium molybdate and 6 drops of stannous chloride to 5.00 mL of each solution. You’ll need to wait 5 minutes for the complex to form before collecting your data. Take measurements from the least concentrated to the most concentrated using the red LED. Don’t forget to include a blank.

When you go to test your water sample, you’ll need to dilute it 50-fold. How much of your water sam-ple will you need to do this? How much distilled water should you add?

Create a standard curve by graphing absorbance vs. concentration by saving your data on the

MeasureNet station using “Manual Entry.” Save the file as “001.” You will want to display the linear regression equation and the r-squared values on your final graph.

Nitrate DeterminationThe Nitrate tests are very similar to those of pH tests, as you will need to dip a small part of the test paper into your solution and compare it with the color chart on the bottle after 30 seconds and 60 seconds. Record all of your observations in your lab notebook.

TurbidityTurbidity measurements are taken in a similar manner to the absorbance readings produced by the colorimeter. Your probe will need to be cali-brated with DI water and 100 NTU solution. Once you have calibrated your instrument, you will need to create a standard curve so you will be able to identify the relationship between your instru-ment’s response to turbidity (the signal) and the known turbidity standards (1 NTU, 10 NTU, and 100 NTU). Don’t forget to shake up the samples before taking your measurements and only use ~3 mL of the solution in your cuvette.

Create a standard curve by graphing the Turbidity Signal versus Turbidity Standard (NTU) using “Manual Entry” on the MeasureNet station. Save the file as “002.”

Use approximately 3 mL of your water sample to measure its turbidity. Record the measurement in your lab notebook.

Total Dissolved SolidsThe amount of total dissolved solids can be mea-sured using conductivity. Conductivity probes that have not been in frequent use need to be soaked in distilled water for 30 minutes prior to use. This may have already been done for you. While poly-ethylene cups are preferred for conductivity mea-surements, as glass will slightly conduct electricity, the error due to this is minimum for our purposes. Any equipment used for this measurement must be cleaned with DI water prior to use to remove any residual ions.

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You will need to calibrate your probe using a 0.01 M KCl solution. Alternatively, a NaCl solution may also be provided. Then you will need to test the conductivity of your water sample.

You will want to produce a standard curve of at least 3 data points, including one of the DI water. You will use less than 3 mL of each solution.

Create a standard curve by graphing the Conductivity versus Concentration, using the “Manual Entry” on the MeasureNet station. Save the file as “003.”

Data AnalysisMake sure to show all of your calculations in your lab notebook as a record of how you completed your calculations. Don’t forget to include your units and correct number of significant figures! Then, go onto Chem21 and report in your results.

1. Identify the site you will use to collect your sample from. Record the GPS coordinates of the place you obtained the sample from, ob-servations of the site, and weather conditions. You may also include a picture of your water site.

Figure 13.5. Hach Nitrate Test Strip Chart.

pH 2. Why is pH important to water quality?

3. What is the EPA Secondary Maximum Contaminant Level for pH?

4. Does your sample fall within the acceptable range?

Temperature5. Record the temperature of the water when you

obtained your sample.

Water Hardness6. Determine the water hardness in terms of mg

CaCO3 per milliliter water for each trial.

7. Report the average and standard deviation for your work.

Phosphate Determination8. Determine the concentration of your phos-

phate standard solutions based upon the vol-umes you measured out and used.

9. View the graph of the known concentration phosphate solutions on Chem21. Using Beer’s law:

a. Determine the slope of the line.

b. Plot the y-intercept.

c. Determine the r2 value.

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These values can be found on your graph. R2 tells us how well the linear regression fits the data points. R2 = 0 means the data points are spread widely around the line, while r2 = 1 means the line fits on the data path (Figure 13.6).

r2 = 0 r2 = 0.99

vs.

Figure 13.6. R2 values for a badly fitting and good fitting best-fit line.

10. What is the actual phosphate concentration of your sample?

Nitrate Determination11. Record the nitrate concentration in mg/L.

12. Using the EPA’s Web site, determine the regu-lated level for nitrates in drinking water.

13. Determine the percent difference between your measurement and the EPA regulated level.

Turbidity14. Graph your standard curve. Determine the

best-fit line and r-squared value.

15. Using your standard curve, determine the tur-bidity of the water sample.

Total Dissolved Solids16. Graph your standard curve. Determine the

best-fit line and r-squared value.

17. Using your conductivity measurement, deter-mine the total dissolved solids.

18. Determine the percent difference between your measurement and the EPA regulated level for dissolved solids.

Water Analysis19. Based on your analysis, would you recom-

mend drinking the water from the water source?

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