Experimental Procedure
A. Preparation of the Spectrophotometer

1. Make sure the spectrophotometer is turned on and let it warm up for at least fifteen minutes before making any measurements.
2. Set the initial wavelength to 360 nm using the buttons on the front of the instrument.
3. Make sure the instrument is set to measure %T (transmittance). You can set the mode using the “mode” button.
4. Adjust the display to 100 %T. The instrument will read BLA—while calibrating. Once the Transmittance value appears, change the mode to Absorbance to record this value in your data.
5. Every time you change the wavelength OR open the top of the spectrophotometer you MUST put the settings back to %T and calibrate to 100% T with your blank solution.
6. The instrument can hold the blank AND 3 solutions at a time. The blank should go in the first space (lever pushed all the way into the instrument), the samples can go in the other 3 spots. When placing the cuvettes into the instrument, they should only be ~ 1⁄2 full and should have a transparent side from left to right in the instrument. Otherwise the light will not be able to go through the sample.

Absorption Spectra of Metal Cations

1. Obtain several cuvettes and make sure they are clean.
2. Fill one cuvette with deionized water. This will be your “blank.”
3. Fill cuvettes with the metal ion solutions. The FeCl3 will be used to find the absorption spectra of Fe3+, the NiCl2 will be used to find the absorption spectra of Ni2+ and the CuSO4 will be used to find the absorption spectra of Cu2+.
4. Carefully wipe each cuvette with a Kimwipe® to remove any dirt or grease from the outside of the tube. From this point on, handle these tubes only at the top, above the liquid level.
5. Place the blank in the sample holder and close the lid.
6. Adjust the %T value to 100%.
7. Change the mode to measure absorbance. The absorption value for your blank should be close to 0.
8. Pull the lever on the front of the instrument to adjust so that the instrument is now reading the first sample. Record the absorbance value on the data sheet.
9. Repeat until the you have the absorbance for all three metal cations.
10. Increase the wavelength by 20 nm and repeat steps 5-8. Continue doing this until you have values for wavelengths up to 800 nm.
11. Graph the Absorbance vs. Wavelength for all metal cations onto a single graph (If your instructor agrees, this can be done in Microsoft Excel and attached, otherwise include a manual graph in the space provided). Make sure the graph is labeled appropriately.

Quantitative Spectroscopy (Creating a Calibration Curve)

1. The 0.20 M CuSO4 solution used in Part B will be referred to as “Copper Standard 1” 2. Prepare4morecopperstandardsasfollows:

1. Copper Standard 2: Obtain additional 0.20 M CuSO4 solution from the tray. Pipet 10 mL of 0.20 M CuSO4 solution into a 50 mL Erlenmeyer flask. Pipet 10 mL of DI water into the same tube and mix well.
2. Copper Standard 3: Pipet 10 mL of Copper Standard 2 into another 50 mL Erlenmeyer flask. Add 10 mL of DI water and mix well.
3. Copper Standard 4: Pipet 10 mL of Copper Standard 3 into another 50 mL Erlenmeyer flask. Add 10 mL of DI water and mix well.

3. Calculate the concentration of all copper standards made with dilution and record the values in your data section.
4. Transfer samples of copper standards to cuvettes. Fill your last cuvette with a solution of copper sulfate of unknown concentration.
5. Use your data for copper in Part B, find the wavelength with the highest absorbance for copper and copy the data to this section.
6. Set the wavelength on the instrument to 10 nm less than the wavelength with the highest absorbance. Place the blank and copper Standard 1 in the spectrophotometer and set %T to 100% as you did before. Obtain and record the absorbance.
7. Repeat using the wavelength 10 nm higher than the wavelength selected from part A. [For example, if your copper sample had its highest absorbance at 640 nm, you will take new measurements at 630 nm and 650 nm.] If your wavelengths are below 600 nm.
8. Look at the data for your three wavelengths and set the instrument to the wavelength which produced the highest absorbance. Once again place the blank in the chamber and adjust the %T reading to 100%.
9. Record both absorbance and %T values for all five copper solutions. (The four standards and the unknown.) Remember that when you open the instrument you must recalibrate to 100 %T with your blank.

10.The unknown concentration will be determined using a graph (calibration curve).

Pre-lab Assignment/Questions

1.

2.

N o t e – this pre-lab must be finished before you come to lab. (Please see syllabus for how to submit this assignment.)

Explain why “roses are red.”

You have a sample of unknown concentration. The species you are evaluating has a molar absorbtivity is 11.02, the path length is exactly 1 centimeter and the absorption is 0.621, calculate the concentration using the equation A = εbc.

3.

You create a calibration curve of Absorbance vs. Concentration using a set of standards. You calculate the trendline to be y = 1.23x + 0.024. the 0.024 is the y intercept or the absorbance when the concentration is 0. What could have caused this value to be greater than 0?

4.

Is the molarity of a solution independent of temperature? Why or why not? Give a better concentration value for use. Defend your choice.

Experimental Data and Results:

Part B. Absorption Spectra of Metal Cations
Record the absorbance for the metal cations at the wavelengths.

Wavelength

    360
    380
    400
    420
    440
    460
    480
    500
    520
    540
    560
    580

Cu2+

Fe3+

Ni2+

Wavelength

600 620

640 660

    680
    700
    720
    740
    760
    780
    800

Cu2+ Fe3+ Ni2+

Attach a graph of absorbance vs. wavelength using the data for all four solutions. (Absorbance should be on the y-axis.) Clearly label the graph. Include a legend and titles for your graph.

Experimental Data and Results: Part C. Quantitative Spectroscopy

Wavelength used for Analysis in Part C: ___________________

*Show an example calculation obtaining the molarity of the standards.

Before continuing, clean all equipment and glassware. Return all materials to their proper location, and turn off the spectrophotometer. Check this box when this is done and all items in your drawer are accounted for.

Instructor Signature_______________________________________________________________

**Using the absorbance graph on the next page you can determine the concentration of the unknown. Take the absorbance value for the unknown and find that value on the y-axis. Draw a horizontal line from that point on the y-axis to the plotted line. From the point where the horizontal line intersects the plotted line, draw a vertical line straight down to the x-axis. Record the value from the point where you hit the x-axis as the unknown concentration in the table.

Experimental Data and Results

Prepare a graph of Transmittance vs. Concentration using the data for the copper standards. Create it in the space provided or in Microsoft Excel and attach in the space provided (depending on your instructor’s directions).

Prepare a graph of Absorbance vs. Concentration using the data for the copper standards. Create it in the space provided or in Microsoft Excel and attach in the space provided (depending on your instructor’s directions).

Post Lab Questions

*See this syllabus for instructions on how to turn in this section of the lab handout.

1. The presence of a dirty fingerprint on the cuvette during measurement of the sample solution resulted in the absorbance being reported incorrectly. Do you think the number reported was too high or too low? Explain why.

2. List the important sources of error in this experiment and what effect each would have on the results. Discuss in particular any errors that you may have made. Include at least 3 (with specific implications) for full credit.

3. A student makes a solution for analysis by mixing 5.172 grams of Cu2SO4 into 500. grams of water. The density of water at the temperature of the lab at the time the solution was made is 0.9926 g/mL. Calculate the molarity, molality, mole fraction, and mass percent of the copper I sulfate. Show all work.