Volumetric or gravimetric determination of ions can be quite complicated and time consuming. At times ion concentrations are too low to be determined with accuracy. When this occurs chemists will consider using an instrument that measures the quantity of light energy that is absorbed by dissolved ions as light is passed through the solution. A colorimeter or spectrophotometer can be used as the tool to determine the concentration of these solutions. If the ions do not produce an intensely colored solution they can sometimes be converted to complex ions that are brightly colored, absorbing light in the visible range. A typical example is Cu²⁺ ion which is converted to the intensely colored Cu(NH₃)₄²⁺ ion by addition of concentrated aqueous ammonia NH₃(aq). The percent transmittance at various concentrations is collected and graphed to determine the concentration of copper(II) in an unknown solution.

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To learn how a spectrophotometer can be used to determine the concentration of a colored solution.

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Wear protective glasses and an apron at all times. Avoid skin contact with the solutions. Avoid inhaling fumes from aqueous ammonia. Dispose of all solutions in the container designated by your teacher. Wash your hands before leaving the laboratory.

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Prepare a data table. Label vertical columns: Test tube #, [Cu²⁺], %ÊT

Preparation of test solutions

1. Obtain eight clean, dry, 50 mL beakers. Label them 1 to 8.
2. Obtain 100 mL of 0.050 M copper(II) sulfate, CuSO₄, stock solution.
3. Obtain two burets. Clean and rinse both burets with distilled water. Fill one cleaned buret with distilled water.
4. Rinse three times with your solution of copper(II) sulfate, using about 10 mL of solution each time. Fill the buret with your copper(II) sulfate solution.
5. Prepare increasingly dilute solutions of copper(II) sulfate as shown below: (In each instance swirl the beaker to mix the two liquids thoroughly.)

   Beaker	1	2	3	4	5	6	7	8
   0.50 M CuSO4 (mL)	10.0	8.0	6.0	4.0	2.0	1.0	1.0	1.0
   distilled H2O	0.0	2.0	4.0	6.0	8.0	9.0	19.0	49.0

6. Obtain ten matched test tubes. Label them 1 through 8. Label the ninth one distilled water and the tenth one "unknown".
7. Pour 5 mL of each prepared solution into its corresponding test tube. Pour 5 mL distilled water into Tube 9.
8. Add two drops concentrated ammonia, NH₃(aq), to test Tubes 1 through 8. Stir. (Caution: Avoid inhaling the ammonia; keep the dropper bottle stoppered.)
9. Using instructions for the operation of your spectrophotometer,
   1. set the wavelength to be used.
   2. zero the instrument.
   3. check for 100% transmittance, % T, using Tube 9. Repeat this step before each sample is read. Keep the well covered at all times to prevent stray light from entering the instrument.
10. Place Tubes 1 through 8, one at a time, in the well and read the % T for each solution. Record.
11. Obtain an unknown solution containing copper(II) ion. Pour 5 mL into Tube 10. Add two drops concentrated aqueous ammonia. Read the % T for this unknown.
12. Dispose of all solutions in the marked container. Wash all glassware. Rinse with distilled water.
13. Wash hands thoroughly before leaving the laboratory.

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1. Calculate the concentration of Cu(NH₃)₄²⁺ion in each test tube. (For example, Tube 2: 8 mL/10 mL x 0.050 M = 0.040 M) Record these calculated values on your data chart.
2. Plot a graph of % T vs. concentration Cu(NH₃)₄²⁺, plotting % T on the vertical axis.
3. What does this graph tell you about the relationship between percent transmittance and concentration?
4. If you need more data to produce a smooth curve prepare additional concentrations of the copper(II) ion. Measure the % T and add these values to your graph.
5. Readings above 95% T are unreliable. From your graph determine the lowest concentration limit of Cu(NH₃)₄²⁺ ion detectable by this procedure.
6. Using the graph just completed and the % T reading for your unknown, determine the unknown copper(II) ion concentration.

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1. A sample of contaminated water is suspected to contain copper(II) ions. Addition of NH₃(aq) does not produce the typical deep blue color of the Cu(NH₃)₄²⁺ ion. The spectrophotometer reads near 100% T. Does this mean that absolutely no copper(II) ions are present? Describe a procedure to determine the amount of Cu²⁺ ion in a very dilute solution. With your teacher's approval, try your procedure.
2. A solution tested for copper(II) ion has an spectrophotometer reading of 0% T. How could you decide whether the concentration reading from the graph is equal to or greater than, the actual concentration in this solution? Describe a procedure to determine the Cu²⁺ ion in such a very concentrated solution. With your teacher's approval, try your procedure.
3. Try answering these items without notes. Then check yourself.
   1. How does a spectrophotometer work? What are the main parts? (See the instruction manual.)
   2. Why is it necessary to set the instrument initially to 100% T? Explain why "blank" is used.
   3. What is the most difficult step in using this instrument?
4. What does solution concentration mean?
5. What do you think the role of a spectrophotometer might be in chemical research?

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1. There is concern about an increasing amount of copper(II) ion in drinking water.
   1. Use the Readers Guide to Periodic Literature in the library to find recent articles on this topic.
   2. Find out what commercial products contain copper(II) ions. For example, visit the local hardware or seed store. Read labels on such items as fertilizers, herbicides, pesticides, algaecides, etc.
   3. Cite references for your accumulated information. Write a conclusion regarding what you have learned about copper(II) ions in drinking water.
   4. CuSO₄•5H₂O is added to swimming pools and ponds to control algae. Bring in a water sample from a local pool or treated pond. Run a test for copper(II) ion.
   5. Gather samples of runoff water from fertilized fields, drainage ditches, or wherever copper(II) ion pollution might be found. Analyze the samples.
2. For Advanced Chemistry: Using Beer's Law and % T data calculate absorbance values for Samples 1 through 8, and plot a graph of absorbance vs. [Cu²⁺]. Explain the difference in the shape of the curve from your plot of % T vs. concentration.

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Teachers Guide

Preparing for the Laboratory Activity

• Major Chemical Concept
• Level
• Expected Student Background
• Time
• Safety
• Materials
• Advance Preparation

Conducting the Laboratory Activity

• Pre-lab Discussion
• Teacher demonstration
• Teacher/Student Interaction
• Anticipated Student Results
• Answers to Data Analysis
• Answers to Imply, Apply
• Possible Extensions
• Post-Lab Discussion
• Possible Extensions

Assessing the Laboratory Learning

• Paper/pencil Items
• Laboratory Practical
• Answers to Pencil/Paper Items
• Answers to Laboratory Practical

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Preparing for the Laboratory Activity

Major Chemical Concept

The concentration of a dilute, colored solution can be determined by an instrument that measures the percent transmittance of light through the solution.

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General and advanced chemistry

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Students should be able to:

• Use burets
• Complete molarity and dilution calculations
• Operate a spectrophotometer
• Prepare and interpret graphs

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Day 1: 50 min

Day 2: 50 min (if extensions are completed)

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• Concentrated aqueous ammonia and copper sulfate are toxic. Do not get them on skin. Do not inhale fumes from aqueous ammonia.
• Remind students of safety precautions when filling burets.

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Non-Consumables (For each four lab teams)

• 1 colorimeter, visible spectrophotometer, or a computer interfaced unit with a green L.E.D. Suggested interface systems: Project SERAPHIM blocktronics or HRM Software Experiments in Colorimetry. Depending on the unit selected, you may need to use a less concentrated solution than used is specified in this activity.
• 8 Beakers, 50 mL
• 1 Buret stand
• 2 Burets, 50 mL
• 1 Stirring rod, glass
• 10 test tubes, matched, to fit in instrument well

Consumables

• 0.050 M copper(II) sulfate, CuSO₄, stock solution, 12.48 g
• CuSO₄•5H₂O per liter solution. Check your spectrophotometer for the optimum concentration to use. Each team will need 100 mL of stock solution.
• NH₃(aq), concentrated, in dropping bottle
• Samples of unknown concentration of copper(II) ion. One 5 mL sample per student, each within range of graph.

For student Imply, Apply items:

• 5 mL sample per student ; [Cu²⁺] less than 0.0010 M
• 5 mL sample per student; [Cu²⁺] greater than 0.050 M

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1. Prepare sets of matched test tubes. Use test tubes from a new box. Fill each with distilled water and check the percent transmittance of each at a wavelength such as 450 nm. Make sets of those with the same % T. Commercial cuvettes can be used in place of the test tubes.
2. Determine the optimum wavelength to be used on the instrument using the stock solution. ( around 460 nm).
3. First set of unknowns: [Cu²⁺] should be within the limits of the graph (0.0020 M to 0.050 M).
4. Second set of unknowns: [Cu²⁺] should be less than 0.0010 M or exceed 0.050 M.

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Conducting the Laboratory Activity

Pre-lab Discussion

This activity must be preceded by an explanation of how the instrument works and how to operate it.

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Use a colored solution to demonstrate the change of percent transmittance (% T) with dilution. Suggested solutions: Kool Aid₂, grape juice, or food coloring. You will need to find the optimum wavelength.

The discussion can be expanded to include Beers' Law and the determination of absorbance. This is not needed for the basic laboratory activity. It would be appropriate for an advanced chemistry class.

A review of dilution calculations would be useful.

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Circulate in the laboratory to help with dilutions if needed.

Be available near the spectrophotometer. The 100% T setting tends to drift. Students may need to be reminded to check it with the blank before each sample reading.

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Sample Data at wavelength 460 nm, using colorimeter

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1. Calculations for dilutions in beakers.

   Beaker 2 8.0 mL/10.0 mL x 0.050 M = 0.040 M

   Beaker #	3	4	5	6	7	8
   CuSO4 (M)	0.030	0.020	0.010	0.005	0.0025	0.0010

2. Typical graph for % T vs. [Cu²⁺]:

   

3. As the concentration decreases the % T increases.
4. Allow students to prepare and analyze dilutions within reason.
5. Value will depend on student graph.
6. Concentration reported by students should correspond within ⁺ 0.01 to the concentration prepared by you.

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1. If the instrument records 100 % T there may be no copper(II) in solution or the concentration of the ion might be less than the level that can be detected by the instrument.

   Response to question of how to determine the concentration would be to evaporate a known quantity of the solution until a very pale blue color appears. Measure the new volume, then test again.

2. If the reading is 0% T the solution probably has more copper(II) in solution than can be measured by the instrument. The solution could be diluted by a measured amount and a new test made.

   For Trials for 1 and 2: Give the student an unknown in which the copper(II) ion is less than or exceeds the limitations of the instrument to analyze. Students should be graded, primarily, on their procedure rather than on their accuracy.

3. The working of the spectrophotometer should include: the light source, the monochrometer, (probably a diffraction grating to shield out all but a small range of wavelengths), the sample holder (all test tubes must be alike), the detector, (probably a phototube to collect the transmitted light), the amplifier (used to increase signal to be fed to the readout device.), the readout dial, (usually in % T).
   1. A selected band of wavelengths (light energy) is allowed to pass through a sample. A portion of light energy is absorbed by the colored ions in the solution depending on their concentration. The remaining light energy passes on through the sample and is detected by a phototube. If the instrument is calibrated properly it records the ratio of the light energy emerging from the sample to the intensity of light energy originally entering the sample. This is recorded as % T.
   2. Water and glass absorb some light energy. This has to be taken into account. The blank must include the same glass and the same solvent.
   3. The most difficult step is the preparation of the test solutions to produce the graph.
4. Solution concentration is given as mol/L. In the case of CuSO₄(aq), this indicates how many colored ions are present in a given volume of solution.
5. This instrument when properly calibrated can save time when an analysis of the concentration of colored ions in a solution is needed. This is especially true when the ion concentration is very dilute.

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1. Student library research should include reviews of articles read, data obtained during trips to the local stores and the concluding paragraph.
2. Students must refer to Beers' Law and calculate absorbance before a graph is drawn. Referring to the equations one is a direct proportion, the other is a logarithmic relationship. Therefore % T vs. concentration is a curved line while absorbance vs. concentration is a straight line.
3. A further extension of this process involves interfacing a computer with a colorimeter to record and process the data directly.

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This discussion should include student difficulties in dilution or calculations, comparing the use of the instrument to determine [Cu²⁺] with "wet chemistry" methods in terms of time, accuracy, and ease of operation, discussing the toxicity of Cu²⁺ and sharing the results of investigations of commercial uses of Cu²⁺.

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1. A good Science Fair project could be developed using the student extension involving samples of pond water or runoff water containing copper(II) ions.
2. If students have had practice in diagraming particulate nature of matter, consider the following:

Draw a beaker 1/4 full of water with Cu(NH₃)₄²⁺ ions in the water. (Use 12 circles to represent the complex ions. Omit the spectator ions and the water molecules.) Draw two more beakers with the water level increased to 1/2 full, to full. What happens to the complex ions in Beakers 2 and 3? (Hopefully the students will scatter the same number of complex ions in the larger volumes of water showing "homogeneity" of the solutions.) Draw a box representing the monochrometer slightly to the left of each beaker. (Students should draw six horizontal lines representing the radiation leading from the monochrometer to the beakers. See Figure T18V2.) Assuming 50% T in Beaker 1 extend three of the lines to the right of the beaker. Use the graph to determine what percent of the radiation passing through Beakers 2 and 3.

If Beaker 1 shows 50% T, with three lines emerging, then Beaker 2 should be about 66% T, with four lines emerging.

Beaker 3 should be about 78% ,T, with five lines emerging.

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Assessing the Laboratory Learning

Paper/pencil Items

1. Draw a schematic illustrating the operation of a colorimeter or a spectrophotometer.
2. If a sample of copper(II) sulfate to be tested contains ethanol, 50% by volume, what changes, if any, would have to be made in the procedure you followed?
3. Use your laboratory graph to determine the concentration of a solution of copper(II) ion with a reading of 40% T.
4. Dora, Gloria, and Mary each took a sample of "run-off" water from a fertilized field in which pesticides containing copper(II) ion had been used. They had previously developed a graph for [Cu²⁺] vs. % T.
   • Gloria measured out three 10 mL samples, added two drops of concentrated aqueous ammonia to each. She turned on the spectrophotometer and read the % T for each sample. She referred to the graph and recorded the [Cu²⁺].
   • Mary poured some of her sample into each of three test tubes and added two drops of concentrated aqueous ammonia to each. She set the wavelength absorbed by Cu(NH₃)₄²⁺. She checked the 0% T and 100% T. Then she read the % T of her three samples. She referred to the graph and recorded the [Cu²⁺].
   • Dora poured her sample in three test tubes, added 8.0 M HCl until a yellow green color appeared, showing the presence of CuCl₄^2- ion. She pushed Mary aside and used Mary's settings to read the % T of her samples.
     • Critique the laboratory technique of each student: Dora, Gloria, and Mary.
5. Anthony collected a sample of water from his swimming pool after some copper(II) sulfate had been added to control green algae. He measured a sample and added concentrated aqueous ammonia. No blue color was observed. Using proper technique, he calibrated his colorimeter. With his sample in place the instrument registered 100% T. Suggest how Anthony might be able to find the copper(II) ion concentration in his pool.

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Each team of four students is given a solution of a cation of unknown concentration to analyze. Solutions are Co(H₂O)₆²⁺, or Ni(H₂O)₆²⁺. Stock solutions, 0.050 M , of each of the cations will be provided. Dropping bottles of concentrated NH₃(aq) are provided. A list of wavelength settings should be prepared by the teacher before the activity. These must be requested by the students when they are ready to use the Spectrophotometer. The teams are to develop their procedure patterned after the activity involving Cu(NH₃)₄²⁺. Members of the team should divide up the activities equally. A written procedure must be approved by the teacher before the team can begin to work in the laboratory.

Teacher Check List per team should include:

1. Team effort to prepare the activity
2. Completeness of the written laboratory procedure
3. Technique in preparing dilutions
4. Proper labeling of beakers and test tubes
5. Requested wavelength
6. Set 0% T of spectrometer
7. Set 100% T using blank
8. Clean up of instrument area
9. Proper disposal of solutions
10. Cleanup of laboratory counter
11. Calculations of dilution concentrations
12. Graph prepared properly
13. Concentration of unknown determined

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1. See below.

   

2. If alcohol is present: A 50% by volume of ethanol and water should be prepared as a blank before the 100% transmittance is adjusted.
3. Answer from their graph.
4. Gloria: She did not set the wavelength, the 0% T, nor the 100% T. She did analyze three samples rather than relying on one to be correct. Her results were useless. She could have corrected the procedure by properly calibrating her instrument and taking new readings.

   Mary: She used proper procedures.

   Dora: She used HCl solution instead of aqueous ammonia. She was rude in pushing Mary aside. The instrument was not calibrated to read % T for [CuCl₄^2-}. The graph was not useful. She used only one sample. Her results were useless. If she had obtained a wavelength setting and a graph for CuCl₄^2- in a previous experiment she could have determined the concentration of her solution. This would have been a good check on Mary's results.

5. Anthony should take a 100 mL sample and boil it down to 10 mL. Allow the sample to cool and run a new test. If the readout is still 100% T Anthony should concentrate a liter of the pool water to, say, 10 mL.

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Teacher must determine the wavelength settings to be used. Stock solutions should be 0.050 M to correspond to the earlier activity. Unknowns must be within the limits of the projected graph. Students should be graded as a team.

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