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This publication may contain or reference information and products protected by copyrights or patents and does not convey any license under our patent rights, nor the rights of others. We do not assume any liability arising out of any infringements of patents or other rights of third parties.

We make no warranty of any kind with regard to this material, including but not limited to the implied warranties of merchantability and fitness for a particular purpose.

Copyright © 2003 by Thermo Electron Corporation, Madison, WI 53711. Printed in the United States of America. All world rights reserved. No part of this publication may be stored in a retrieval system, transmitted, or reproduced in any way, including but not limited to photocopy, photograph, magnetic, or other record, without the prior written permission of Thermo Electron Corporation.

For technical assistance, please contact:

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SPECTRONIC, GENESYS and Educator are trademarks of Thermo Electron Scientific Instruments Corporation, a subsidiary of Thermo Electron Corporation.

332909-10033 Rev. R DISCLAIMER

While every reasonable effort has been made to ensure that you receive a product that you can use and enjoy, Thermo Electron Corporation does not warrant that the function of the product will meet your requirements or that the operation of the product will be uninterrupted or error-free. IN NO EVENT WILL THERMO ELECTRON CORPORATION BE LIABLE TO YOU OR ANY OTHER PARTY FOR DIRECT, INDIRECT, GENERAL, SPECIAL, INCIDENTAL, CONSEQUENTIAL, EXEMPLARY OR OTHER DAMAGES ARISING FROM THE USE OF OR INABILITY TO USE THE PRODUCT OR FROM ANY BREACH OF ANY WARRANTY, EVEN IF THERMO ELECTRON CORPORATION HAS BEEN ADVISED OF THE POSSIBILITY OF SUCH DAMAGES. (Some states do not allow the exclusion or limitation of incidental or consequential damages, so that above exclusion or limitation may not apply to you.) In no event shall the total liability of Thermo Electron Corporation exceed the amount you paid for the right to use this product. Because it is impossible for Thermo Electron Corporation to know the purposes for which you acquired this product or the uses to which you will put this product, you assume full responsibility for the selection of the product and for its use and the results of that use. ACKNOWLEDGEMENT

Thermo Electron Corporation wishes to thank Professors Robert Smith and Michael Walczak of the Chemistry Department at St. John Fisher College in Rochester, New York, for rewriting this manual. We also extend our thanks to Linda Mocejunas and Beth Reabold at St. John Fisher College for their help in typing and formatting the experiments, and to the chemistry students of St. John Fisher College and those high school students from the Rochester, New York, area who tested these experiments and provided constructive feedback.

Thermo Electron Corporation INTRODUCTION

The experiments in this manual have been selected to provide high school and/or first-year college students with a variety of experiences using visible spectroscopy. Experiments 1-4 are basic experiments. Experiments 1 and 7 each involve building a spectrophotometer and then investigating how the instrument operates. Experiments 2-4 provide an introduction to spectroscopy: absorption spectra are recorded and the relationship between absorbance and concentration (shown by Beer's Law) is investigated. Experiments 5, 6, and 8-10 illustrate how visible spectroscopy can be employed to solve problems of chemical interest.

Experiments 1-7 can be performed individually or in groups, and the experiments are designed so that all of the experimental data needed to answer the questions posed in the experiments can be collected in approximately one hour of lab time. Furthermore, all of the reagents needed in Experiments 2-5 can be purchased at a grocery store, and most of the reagents for Experiment 6 can be found at a pet store. Therefore, Experiments 1-7 are well-suited for high school students. Experiments 8-10 are intended to be performed individually, require approximately three hours each to complete, and are not easily broken into parts which could be done simultaneously. While these experiments are intended for college students, they could also be performed by high school students having a sufficiently long lab period.

Each experiment in the lab manual has been written so that it can stand alone. No experiment makes reference to material in any other experiment in the manual. Any of the experiments may be copied for distribution to your students.

The same format is used in each experiment. The “Introduction” states what is to be investigated and provides the background needed for that experiment. The “Experimental Procedure” section has detailed directions for performing the experiment; included in this section are safety warnings and directions for disposal of wastes. The “Calculations” section states specifically the quantities to be calculated and the questions to be answered. Pages for recording the data, showing calculations, and writing the answers to the questions are included in the “Report Form” section. The final section, “Notes to Instructor,” provides information about the amounts of chemicals and materials needed per person (or per group), directions for solution preparations, comments about expected experimental results, and answers to questions. In addition, some “Notes to Instructor” sections have additional information about safety matters and waste disposal. Whenever possible, the experiment uses only small quantities of reagents.

Please read all warnings on the labels on the reagent bottles. The applicable Material Safety Data Sheet (MSDS) can be consulted for additional information, including medical response, about a specific chemical. Always have your students wear safety goggles, or other appropriate eye protection, when they are working in the laboratory.

TABLE OF CONTENTS

PAGE

GENERAL OPERATING INSTRUCTIONS

i. SPECTRONIC 20 SPECTROPHOTOMETER ...... i-1

ii. SPECTRONIC 20D SPECTROPHOTOMETER ...... ii-1

iii. SPECTRONIC 20+ SPECTROPHOTOMETER ...... iii-1

iv. SPECTRONIC 20D+ SPECTROPHOTOMETER ...... iv-1

v. SPECTRONIC Educator SPECTROPHOTOMETER ...... v-1

vi. GENESYS 20 SPECTROPHOTOMETER ...... vi-1

EXPERIMENTS

1. Compact Disc (CD) Spectrophotometer ...... 1-1

2. Absorption Spectrum...... 2-1

3. Determination of Concentration Using Beer's Law...... 3-1

4. Importance of Wavelength Selected in the Beer's Law Experiment...... 4-1

5. Red Cabbage pH Indicator...... 5-1

6. Spectrophotometric Determination of Nitrate Ion Concentration ...... 6-1

7. Comparing the Performance of Two Spectrophotometers ...... 7-1

8. Formula Determination by Continuous Variations ...... 8-1

9. Determination of the Solubility Product of Copper (II) Iodate ...... 9-1

10.A Kinetics Experiment: Fading of Phenolphthalein...... 10-1

GENERAL OPERATING INSTRUCTIONS

SPECTRONIC® 20 SPECTROPHOTOMETER

GENERAL

1. Turn on the SPECTRONIC 20 by turning the power switch clockwise. Allow the spectrophotometer to warm up for at least fifteen minutes to stabilize the source and detector.

2. Set the desired wavelength with the wavelength control.

3. Adjust the meter to 0%T with the zero control knob.

4. Fill a clean cell with water (or another blank solution) and wipe the cell with a tissue to remove liquid droplets, dust and finger prints. Place the cell in the sample compartment and align the guide mark on the cell with the guide mark at the front of the sample compartment. Press the cell firmly into the sample compartment and close the lid. Adjust the meter to 100%T with the transmittance/absorbance control knob. Remove the cell from the sample compartment and empty the water.

5. When all measurements are completed, turn off the spectrophotometer by turning the power switch counterclockwise until it clicks.

MAKING ONE OR MORE MEASUREMENTS AT THE SAME WAVELENGTH

1. Follow steps 1 through 4 above (“General”).

2. Rinse the cell twice with small volumes of the solution to be measured and fill it with the solution. Wipe the cell with a tissue and insert the cell into the sample compartment. Align the guide marks and close the lid. Read the appropriate value (%T or A) from the meter.

3. Remove the cell from the sample compartment and repeat step 2 for any remaining sample solutions.

i-1 GENERAL OPERATING INSTRUCTIONS SPECTRONIC 20 SPECTROPHOTOMETER

MAKING MEASUREMENTS AT MORE THAN ONE WAVELENGTH

NOTE: If you need to take measurements at many wavelengths, it may be more convenient to use separate cells for the blank and for the sample.

1. Follow steps 1 through 4 above (“General”) for the first wavelength.

2. Rinse the cell twice with small volumes of the solution to be measured and fill it with the solution. Wipe the cell with a tissue and insert the cell into the sample compartment. Align the guide marks and close the lid. Read the appropriate value (%T or A) from the meter.

3. Repeat steps 1 and 2 for each new wavelength.

NOTE: It is important to realize that the response of the instrument changes with wavelength. You must reset the meter to 100%T every time the wavelength is changed.

i-2 GENERAL OPERATING INSTRUCTIONS

SPECTRONIC 20D SPECTROPHOTOMETER

GENERAL

1. Turn on the SPECTRONIC 20D by turning the power switch clockwise. Allow the spectrophotometer to warm up for at least fifteen minutes to stabilize the source and detector.

2. After the warmup period, set the desired wavelength with the wavelength control.

3. Set the display mode to “transmittance” by pressing the mode control until the LED beside “transmittance” is lit.

4. Adjust the display to 0%T with the zero control knob.

5. Fill a clean cell with water (or another blank solution) and wipe the cell with a tissue to remove liquid droplets, dust and finger prints. Place the cell in the sample compartment, align the guide mark on the cell with the guide mark at the front of the sample compartment. Press the cell firmly into the sample compartment and close the lid. Adjust the display to 100%T with the transmittance/absorbance control. Remove the cell from the sample compartment and empty the water.

6. When all measurements are completed, the spectrophotometer can be turned off by turning the power switch counterclockwise until it clicks.

MAKING ONE OR MORE MEASUREMENTS AT THE SAME WAVELENGTH

1. Follow steps 1 through 5 above (“General”).

2. Rinse the cell twice with small volumes of the solution to be measured and fill it with the solution. Wipe the cell with a tissue and place the cell in the sample compartment. Align the guide marks and close the lid. You can read %T directly from the display. To read absorbance, press the mode control switch until the LED beside “absorbance” is lit.

3. Remove the cell from the sample compartment and repeat step 2 for any remaining sample solutions.

ii-1 GENERAL OPERATING INSTRUCTIONS SPECTRONIC 20D SPECTROPHOTOMETER

MAKING MEASUREMENTS AT MORE THAN ONE WAVELENGTH

NOTE: If you need to take measurements at many wavelengths, it may be more convenient to use separate cells for the blank and for the sample.

1. Follow steps 1 through 5 above (“General”) for the first wavelength.

2. Rinse the cell twice with small volumes of the solution to be measured and fill it with the solution. Wipe the cell with a tissue and place the cell in the sample compartment. Align the guide marks and close the lid. You can read %T directly from the display. To read absorbance, press the mode control switch until the LED beside “absorbance” is lit.

3. Repeat steps 1 and 2 for each new wavelength.

NOTE: It is important to realize that the response of the instrument changes with wavelength. You must reset the display to 100%T every time the wavelength is changed.

MAKING CONCENTRATION MEASUREMENTS AT ONE WAVELENGTH

1. Follow steps 1 through 5 above (“General”).

2. Rinse the cell twice with small volumes of the standard solution of known concentration and fill the cell with the solution. Wipe the cell with a tissue and place the cell in the sample compartment. Align the guide marks and close the lid.

3. Press the mode switch until the LED beside “concentration” is lit. Press the increase or decrease switches until the displayed concentration matches the concentration of the standard.

4. Remove the standard, and rinse and fill the cell with the sample solution. Wipe the cell with a tissue and place the cell in the sample compartment. Read the concentration of the sample directly from the display.

5. Repeat step 4 for each of the samples.

ii-2 GENERAL OPERATING INSTRUCTIONS

SPECTRONIC 20+ SPECTROPHOTOMETER

GENERAL

1. Turn on the SPECTRONIC 20+ by turning the power switch clockwise. Allow the spectrophotometer to warm up for at least fifteen minutes to stabilize the source and detector.

2. Set the desired wavelength with the wavelength control.

3. Adjust the meter to 0%T with the zero control knob.

4. Fill a clean cell with water (or another blank solution) and wipe the cell with a tissue to remove liquid droplets, dust and finger prints. Place the cell in the sample compartment and align the guide mark on the cell with the guide mark at the front of the sample compartment. Press the cell firmly into the sample compartment and close the lid. Adjust the meter to 100%T with the transmittance/absorbance control knob. Remove the cell from the sample compartment and empty the water.

5. When all measurements are completed, turn off the spectrophotometer by turning the power switch counterclockwise until it clicks.

MAKING ONE OR MORE MEASUREMENTS AT THE SAME WAVELENGTH

1. Follow steps 1 through 4 above (“General”).

2. Rinse the cell twice with small volumes of the solution to be measured and fill it with the solution. Wipe the cell with a tissue and insert the cell into the sample compartment. Align the guide marks and close the lid. Read the appropriate value (%T or A) from the meter.

3. Remove the cell from the sample compartment and repeat step 2 for any remaining sample solutions.

iii-1 GENERAL OPERATING INSTRUCTIONS SPECTRONIC 20+ SPECTROPHOTOMETER

MAKING MEASUREMENTS AT MORE THAN ONE WAVELENGTH

NOTE: If you need to take measurements at many wavelengths, it may be more convenient to use separate cells for the blank and for the sample.

1. Follow steps 1 through 4 above (“General”) for the first wavelength.

2. Rinse the cell twice with small volumes of the solution to be measured and fill it with the solution. Wipe the cell with a tissue and insert the cell into the sample compartment. Align the guide marks and close the lid. Read the appropriate value (%T or A) from the meter.

3. Repeat steps 1 and 2 for each new wavelength.

NOTE: It is important to realize that the response of the instrument changes with wavelength. You must reset the meter to 100%T every time the wavelength is changed.

iii-2 GENERAL OPERATING INSTRUCTIONS

SPECTRONIC 20D+ SPECTROPHOTOMETER

GENERAL

1. Turn on the SPECTRONIC 20D+ by turning the power switch clockwise. Allow the spectrophotometer to warm up for at least fifteen minutes to stabilize the source and detector.

2. After the warm-up period, set the desired wavelength with the wavelength control.

3. Set the display mode to “transmittance” by pressing the mode control until the LED beside “transmittance” is lit.

4. Adjust to 0%T.

5. Fill a clean cell with water (or another blank solution) and wipe the cell with a tissue to remove liquid droplets, dust and finger prints. Place the cell in the sample compartment, align the guide mark on the cell with the guide mark at the front of the sample compartment. Press the cell firmly into the sample compartment and close the lid. Adjust the display to 100%T with the transmittance/absorbance control. Remove the cell from the sample compartment and empty the water.

6. When all measurements are completed, the spectrophotometer can be turned off by turning the power switch counterclockwise until it clicks.

MAKING ONE OR MORE MEASUREMENTS AT THE SAME WAVELENGTH

1. Follow steps 1 through 4 above (“General”).

2. Rinse the cell twice with small volumes of the solution to be measured and fill it with the solution. Wipe the cell with a tissue and place the cell in the sample compartment. Align the guide marks and close the lid. You can read %T directly from the display. To read absorbance, press the mode control switch until the LED beside “absorbance” is lit.

3. Remove the cell from the sample compartment and repeat step 2 for any remaining sample solutions.

iv-1 GENERAL OPERATING INSTRUCTIONS SPECTRONIC 20D+ SPECTROPHOTOMETER

MAKING MEASUREMENTS AT MORE THAN ONE WAVELENGTH

NOTE: If you need to take measurements at many wavelengths, it may be more convenient to use separate cells for the blank and for the sample.

1. Follow steps 1 through 4 above (“General”) for the first wavelength.

2. Rinse the cell twice with small volumes of the solution to be measured and fill it with the solution. Wipe the cell with a tissue and place the cell in the sample compartment. Align the guide marks and close the lid. You can read %T directly from the display. To read absorbance, press the mode control switch until the LED beside “absorbance” is lit.

3. Repeat steps 1 and 2 for each new wavelength.

NOTE: It is important to realize that the response of the instrument changes with wavelength. You must reset the display to 100%T every time the wavelength is changed.

MAKING CONCENTRATION MEASUREMENTS AT ONE WAVELENGTH

1. Follow steps 1 through 4 above (“General”).

2. Rinse the cell twice with small volumes of the standard solution of known concentration and fill the cell with the solution. Wipe the cell with a tissue and place the cell in the sample compartment. Align the guide marks and close the lid.

3. Press the mode switch until the LED beside “concentration” is lit. Press the increase or decrease switches until the displayed concentration matches the concentration of the standard.

4. Remove the standard, and rinse and fill the cell with the sample solution. Wipe the cell with a tissue and place the cell in the sample compartment. Read the concentration of the sample directly from the display.

5. Repeat step 4 for each of the samples.

iv-2 GENERAL OPERATING INSTRUCTIONS

SPECTRONIC Educator SPECTROPHOTOMETER

GENERAL

1. Turn on the SPECTRONIC Educator by turning the power switch, located on the left front of the instrument, clockwise. Allow the spectrophotometer to warm up for at least fifteen minutes to stabilize the source and detector.

2. Set the desired wavelength with the wavelength control.

3. Set the display mode to "transmittance" by pressing the %T/A rocker switch upward.

4. Fill a clean cell with water (or another blank solution) and wipe the cell with a tissue to remove liquid droplets, dust and finger prints. Place the cell in the sample compartment and align the guide mark on the cell with the guide mark at the front of the sample compartment. Press the cell firmly into the sample compartment and close the lid. Adjust the LCD display to 100%T with the transmittance/absorbance control knob. Remove the cell from the sample compartment and empty the water.

5. When all measurements are completed, turn off the spectrophotometer by turning the power switch counterclockwise until it clicks.

MAKING ONE OR MORE MEASUREMENTS AT THE SAME WAVELENGTH

1. Follow steps 1 through 4 above (“General”).

2. Rinse the cell twice with small volumes of the solution to be measured and fill it with the solution. Wipe the cell with a tissue and insert the cell into the sample compartment. Align the guide marks and close the lid. Read the appropriate value (%T or A) from the display.

3. Remove the cell from the sample compartment and repeat step 2 for any remaining sample solutions.

v-1 GENERAL OPERATING INSTRUCTIONS SPECTRONIC EDUCATOR SPECTROPHOTOMETER

MAKING MEASUREMENTS AT MORE THAN ONE WAVELENGTH

NOTE: If you need to take measurements at many wavelengths, it may be more convenient to use separate cells for the blank and for the sample.

1. Follow steps 1 through 4 above (“General”) for the first wavelength.

2. Rinse the cell twice with small volumes of the solution to be measured and fill it with the solution. Wipe the cell with a tissue and insert the cell into the sample compartment. Align the guide marks and close the lid. Read the appropriate value (%T or A) from the display.

3. Repeat steps 1 and 2 for each new wavelength.

NOTE: It is important to realize that the response of the instrument changes with wavelength. You must reset the display to 100%T every time the wavelength is changed.

v-2 GENERAL OPERATING INSTRUCTIONS

GENESYS 20 SPECTROPHOTOMETER

GENERAL

1. Turn on the GENESYS 20 by pushing the “*” on the On/Off switch, which is located at the rear of the instrument near the A/C power connector. The GENESYS 20 automatically performs its power-on sequence, which includes checking the software revision and initializing the filter wheel and monochromator. The power-on sequence takes about two minutes to complete. Allow the instrument to warm up for 30 minutes before taking measurements.

2. Absorbance and %Transmittance measurements:

a. Press A/T/C to select the absorbance or %transmittance mode. The current mode appears on the display.

b. Press nm • or nm – to select the wavelength. NOTE: Holding either key will cause the wavelength to change more quickly.

c. Insert your blank into the cell holder and close the sample door. NOTE: Position the cell so the light (indicated by arrow in drawing) passes through the clear walls.

d. Press 0 ABS/100%T to set the blank to 0A or 100%T.

e. Remove your blank and insert your sample into the cell holder. The sample measurement appears on the LCD display.

vi-1 GENERAL OPERATING INSTRUCTIONS GENESYS 20 SPECTROPHOTOMETER

3. Concentration measurements using a factor:

a. Press A/T/C to select the concentration mode. The current mode appears on the display.

b. Press nm • or nm – to select the wavelength. NOTE: Holding either key will cause the wavelength to change more quickly.

c. Press the Factor soft key and use the • and – keys to select the factor, then press ACCEPT to accept it.

d. Insert your blank into the cell holder and close the sample door. NOTE: Position the cell so the light (indicated by arrow in drawing) passes through the clear walls.

e. Press 0 ABS/100%T to set the blank to 0 concentration.

f. Remove your blank and insert your sample into the cell holder. The calculated sample concentration appears on the LCD display.

4. Concentration measurements using a standard:

a. Press A/T/C to select the concentration mode. The current mode appears on the display.

b. Press nm • or nm – to select the wavelength. NOTE: Holding either key will cause the wavelength to change more quickly.

c. Insert your blank into the cell holder and close the sample door. NOTE: Position the cell so the light (indicated by arrow in drawing) passes through the clear walls.

d. Press 0 ABS/100%T to zero the blank, then remove the blank and insert your standard.

vi-2 GENESYS 20 SPECTROPHOTOMETER GENERAL OPERATING INSTRUCTIONS

e. Press the Standard soft key and use the • and – keys to enter the concentration of the standard; then press the Set C soft key to calculate and display the factor for the selected standard.

f. Remove the standard and insert your sample into the cell holder. The calculated sample concentration appears on the LCD display.

4. When all measurements are completed, turn off the spectrophotometer by pushing the “F” on the On/Off switch.

MAKING ONE OR MORE MEASUREMENTS AT THE SAME WAVELENGTH

1. Follow the appropriate steps above for the desired mode of operation (“General”).

2. Rinse the cell twice with small volumes of the solution to be measured and fill it with the solution. Wipe the cell with a tissue and insert the cell into the sample compartment. Align the guide marks and close the lid. Read the appropriate value from the display.

3. Remove the cell from the sample compartment, and repeat step 2 for any remaining sample solutions.

MAKING MEASUREMENTS AT MORE THAN ONE WAVELENGTH

NOTE: If you need to take separate measurements at many wavelengths, it may be more convenient to use separate cells for the blank and sample.

1. Follow the appropriate steps above for the desired mode of operation (“General”).

2. Rinse the cell twice with small volumes of the solution to be measured and fill it with the solution. Wipe the cell with a tissue and insert the cell into the sample compartment. Align the guide marks and close the lid. Read the appropriate value from the display.

3. Repeat steps 1 and 2 above for each new wavelength.

NOTE: As the response of the instrument changes with wavelength, you must reset the blank to 0A (or 100%T) each time the wavelength is changed.

vi-3 GENERAL OPERATING INSTRUCTIONS GENESYS 20 SPECTROPHOTOMETER

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vi-4 EXPERIMENT 1

COMPACT DISC (CD) SPECTROPHOTOMETER

INTRODUCTION

A spectrophotometer has five basic parts: a light source, a device that separates the light into its constituent wavelengths, a compartment to hold the sample, a detector that analyzes the light which has passed through the sample, and a readout device to provide data to the instrument's user. In this experiment you will build a simple spectrophotometer using a compact disc to separate white light into its color components. A flashlight is used as the light source and your eye is used as the detector. Building a spectrophotometer should increase your understanding of how the instrument works and the decisions that must be made in designing and building a commercial instrument.

EXPERIMENTAL PROCEDURE

Always wear safety goggles while performing the experiment.

Equipment Needed

Compact Disc (CD) Flashlight or 35 mm projector Aluminum foil 2 pencils or 2 ball point pens 2 large plastic bottles (2-L soda bottles or 1-gal milk containers) Tape Knife, pin, sewing needle, or other similarly sharp object Book or wooden block

Hold the CD near a light source, slightly rotate it, and observe the colors produced when the light reflects off the surface of the CD. You have probably done this many times before. The ability of the CD to separate light striking it into colors means a CD can be used as the device to separate light into its constituent wavelengths in the spectrophotometer you will build.

Let's begin building your spectrophotometer. Place the CD on the table with the label side up. Tape the two pens or pencils in place as shown in the diagram at the top of the next page. Lift the CD above the table top so that the pens or pencils are parallel to the table top, the CD is now perpendicular to the table top, and the bottom edge of the CD is about an inch above the table top.

1-1 COMPACT DISC (CD) SPECTROPHOTOMETER EXPERIMENT 1

Stand two plastic bottles on the table top. Position a bottle by the outer end of each pen or pencil while the bottom edge of the CD is held an inch above the table top. Mark each plastic bottle where a pen points to it. Poke a hole in each bottle at this point. The hole should be large enough to allow the pen to be inserted into the bottle but small enough that the pen fits snugly. Insert the outer end of each pen into its hole; a side view (not drawn to scale) of the set-up is shown in the diagram below. The CD should now be suspended off the surface of the table, and you should be able to rotate the CD about the axis of the pens and leave it in a given position. Rotate the CD so that it is as perpendicular as possible to the table top.

Use a flashlight for your light source. (Alternatively, a 35 mm slide projector can be used as the light source.) Tape the flashlight to a book or block of wood, and position the supported light source near the side of the CD. Cover the flashlight lens with a piece of aluminum foil with a pin hole punched in its center; this gives a narrow beam of light from the flashlight. You want the flashlight to be parallel to the table top, and you want the light beam to be perpendicular to the axis of the pens and to strike the center of the upper half of the CD. This arrangement is called the flashlight-CD alignment and is illustrated in the diagram below. After you have positioned the parts of the instrument and turned on the flashlight to make sure that you have the desired flashlight-CD alignment, be careful not to jar any portion of your set- up; the proper alignment is easily lost.

1-2 EXPERIMENT 1 COMPACT DISC (CD) SPECTROPHOTOMETER

Turn on the flashlight to shine the light beam on the CD, and rotate the CD about the axis of the pens. Do you see any colors where the light beam strikes the CD? If so, what colors do you see? Do the colors change when you rotate the CD? In what order do the colors appear? Are they always in the same order?

Next, place an aluminum foil roof with a tiny hole in it over the CD. This set-up is shown in the following diagram. The purpose of the roof is to allow you to select a small amount of the light reflected off the CD to view at a given time. You can attach the roof to the top of the flashlight or to the plastic bottles. The roof must not touch the CD and must not block the light beam from the flashlight. With this completed, you should be able to look down at the pin hole in the roof over the CD and see one color in the pin hole. Adjust the flashlight-CD alignment until you achieve this.

1-3 COMPACT DISC (CD) SPECTROPHOTOMETER EXPERIMENT 1

Again rotate the CD. Do you still see the colors change? Do the colors appear in the same order as you previously observed without the roof?

You can now try some different samples with your instrument. Try placing some different colored materials over the pin hole. Rotate the CD so different colors of light are passing through your sample. Record your observations. See if you can determine what color light is most absorbed for a given color of material.

COMMENTS

You now have some experience with building and using a spectrophotometer. The table below lists the features of your instrument and the Thermo Spectronic instrument. The ideal light source would produce the same amount of power at all wavelengths, it would produce the same amount of power over time, it would use no power to produce the light, and it would never burn out. The design of the spectrophotometer must consider that the ideal light source does not exist.

The device that breaks the light into its constituent wavelengths is called a monochromator. The ideal monochromator would output the same amount of power that is put into it, allow infinite resolution of wavelengths, allow an infinite range of wavelengths, require no maintenance or calibration, i.e. always give the output that it is set to give, be extremely inexpensive, be constructed of light materials, and be extremely small. When a company designs a spectrophotometer, they must consider that the ideal monochromator does not exist and they must make decisions about the design and construction of the monochromator that trade off between cost and performance.

Feature Your Instrument Thermo Spectronic Instrument Light Source Light Bulb Tungsten Bulb Device to Separate Light Compact Disc Grating Calibrated Wavelength No Yes Selection Sample Compartment No Yes Detector Your Eye Photomultiplier or Solid State Device Output None Meter or Digital Display

Construction Bare Bones Rugged and Attractive

Your instrument has no sample compartment. The Thermo Spectronic instrument has a compartment carefully designed to allow ease of use, hold the sample reproducibly in the light path, avoid contamination of the optics when sample spills occur, and block out external light.

1-4 EXPERIMENT 1 COMPACT DISC (CD) SPECTROPHOTOMETER

The ideal detector would give the same response to an infinite range of wavelengths, be stable over time, require no power, be extremely inexpensive, be small, and last forever. Obviously, this device does not exist, so trade-offs must be made. Earlier Thermo Spectronic instruments used a photomultiplier tube as a detector; some instruments required changing tubes for different wavelength ranges. The 20 "+" instruments have a silicon detector which covers the entire visible range, is smaller, requires less power, and should be more stable with a longer lifetime.

The Thermo Spectronic instruments are built on a rugged chassis to keep the optics aligned and are carefully designed for ease of use by persons learning to use instrumentation. Hopefully, constructing your own spectrophotometer has increased your awareness of how a spectrophotometer works and increased your appreciation of the decisions that go into the design and construction of a commercial instrument.

1-5 COMPACT DISC (CD) SPECTROPHOTOMETER EXPERIMENT 1

COMPACT DISC (CD) SPECTROPHOTOMETER

REPORT FORM

When you rotated the CD before you added the roof, did the colors change? What colors did you see?

In what order do the colors appear? Are they always in the same order?

What colors did you see when you rotated the CD with the roof on? Did the colors appear in the same order as you previously observed?

Sample Observations

1-6 EXPERIMENT 1 COMPACT DISC (CD) SPECTROPHOTOMETER

NOTES TO INSTRUCTOR

The students should work in groups; three per group is good but as many as five per group is acceptable. Use an old CD, preferably one that was to be discarded. Any kind of tape can be used to attach the pens to the CD. You may want to use flat-sided plastic milk cartons to suspend the CD; the cartons may provide a more stable support than do bottles, and it may be easier to make a hole in a flat-sided container. To increase stability, you may also want to add water to the bottles or milk cartons (but do not fill above the holes). Students may become frustrated trying to achieve the proper flashlight-CD alignment, especially when the roof is in place. Use this as an opportunity to discuss why the commercial instrument is built on a solid frame to maintain the alignment of the optics.

Some possible colored samples that can be studied include colored glass, colored cellophane, or any thin, transparent colored plastic. A flashlight does not provide a very intense light beam, so there is little intensity to the beam which is emitted through the pin hole in the roof above the CD. Consequently, you cannot study very thick samples because they would not allow enough light to be transmitted to be observed by the eyes. You may want to use a 35mm projector as the light source, since this gives a more intense beam than does a flashlight.

Students can observe the different colors of light produced by the Thermo Spectronic spectrophotometer by putting a strip of white paper into the sample chamber and observing the color of light at various wavelength settings. If you feel comfortable removing the cover of the Thermo Spectronic instrument, you can show the students the various parts of the instrument and allow them to compare it to the instruments they built. UNPLUG THE SPECTROPHOTOMETER BEFORE REMOVING THE COVER. Do not allow the students to touch the internal components of the Thermo Spectronic instrument.

Materials needed, per spectrophotometer:

Compact Disc (CD) Flashlight or 35 mm projector Aluminum foil 2 pencils or 2 ball point pens 2 large plastic bottles (2-L soda bottles or 1-gal milk containers) Tape Knife, pin, sewing needle, or other similarly sharp object Book or wooden block Colored samples

1-7 COMPACT DISC (CD) SPECTROPHOTOMETER EXPERIMENT 1

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1-8 EXPERIMENT 2

ABSORPTION SPECTRUM

INTRODUCTION

Visible light is that portion of the electromagnetic spectrum which has wavelengths in the range from approximately 380nm to 760nm. If a beam of visible light is shone on a colored solution prepared by dissolving a colored solute in a colorless solvent, the colored component of the solution will typically absorb some of the wavelengths in the light beam and transmit other wavelengths. A spectrophotometer is an instrument which can be used to determine which wavelengths in the visible region are transmitted and which are absorbed by the colored solution. The instrument also can be used to determine the degree or extent of absorption at any wavelength. The degree of absorption is called the absorbance of the solution at that wavelength. A plot of absorbance versus wavelength for a solution is the absorption spectrum for the colored substance in that solution. A wavelength, or a continuous wavelength range, where a maximum in the absorbance value occurs in the absorption spectrum, is called a peak.

In this experiment, you will first record the absorption spectrum over a range of wavelengths in the visible region for each of two stock colored solutions, and determine the wavelengths where peaks occur in each spectrum. You will then dilute one of the stock colored solutions with water, and record the absorption spectrum for this dilute solution, so that you can examine the effect of dilution on the absorption spectrum. Finally, you will prepare an aqueous solution containing the colored substances from both of the stock colored solutions. You will record the absorption spectrum for this solution and use that spectrum to determine the ratio of the volumes of the two stock solutions that were used to prepare the aqueous solution of intermediate color.

EXPERIMENTAL PROCEDURE

Always wear safety goggles while performing the experiment.

Reagents and Equipment Needed

Aqueous solution of red food coloring Aqueous solution of yellow food coloring 10-mL graduated cylinder 2 50-mL beakers 2 cuvettes Spectrophotometer

Rinse one of the cuvettes with distilled water, discard the rinsing in a sink, and fill the cuvette two-thirds full with distilled water. This cuvette will have only distilled water in it for the duration of the experiment. Rinse the other cuvette with several milliliters of the stock aqueous solution of red food coloring (hereafter referred to as the red solution), discard the rinsing in a sink, and fill the cuvette two- thirds full with the red solution. If you spill this solution, or any other solution used in this experiment, on yourself, wash the affected area with a large volume of water. Follow your instructor's directions for the operation of your spectrophotometer, and record the absorbance of the solution from 350nm to

2-1 ABSORPTION SPECTRUM EXPERIMENT 2

540nm at 10nm intervals. Discard the red solution in the cuvette. Rinse the cuvette with two portions of distilled water and discard each rinsing in a sink. Repeat the procedure described above, substituting the stock aqueous solution of yellow food coloring (hereafter referred to as the yellow solution) for the red solution.

Select one of the two stock colored solutions. Rinse the 10-mL graduated cylinder with several milliliters of the solution you selected, and discard the rinsing in a sink. Use the graduated cylinder to transfer 5mL of the colored solution to a clean, dry 50-mL beaker. Use the same graduated cylinder to transfer 5mL of distilled water to the beaker, and swirl to mix the contents of the beaker; this solution is called the diluted stock solution. Repeat the procedure given earlier for collecting the data for the absorption spectrum, substituting the diluted stock solution for the red solution.

Rinse the 10-mL graduated cylinder with several milliliters of the red solution, and discard the rinsing in a sink. Use the graduated cylinder to transfer 3-7mL of red solution to a clean, dry 50-mL beaker; record the volume, to ±0.1 mL, of solution transferred. Use the same graduated cylinder to transfer 3-7mL of yellow solution to the beaker; again record the volume, to ±0.1mL, of solution transferred. You want the sum of the two volumes to be at least 10mL. Swirl to mix the contents of the beaker; this solution is called the orange solution. Repeat the procedure for data collection for the absorption spectrum, substituting the orange solution for the red solution. At the conclusion of the experiment, discard the water in the one cuvette and return both cuvettes.

CALCULATIONS

For each of the four solutions, plot absorbance along the y-axis versus wavelength along the x-axis, and draw a smooth curve through the points for a solution. Plot all absorption spectra on the same piece of graph paper, and indicate the solution to which each spectrum applies. If possible, use a different color for drawing the curve for each spectrum plotted. Use your spectra to determine the wavelength(s) where the peak(s) occur(s) for each colored solution.

Look at the spectra for the stock solution and diluted solution of the same color. Do the peaks occur at the same wavelength? If not, are the wavelengths similar? Are the absorbance values at the peak the same value? What conclusions can you draw about the effect that diluting the stock solution has on the absorption spectrum for the solution? Try to be as specific as possible. If you mixed equal volumes of the diluted stock solution and distilled water, predict the absorbance value you would expect at the peak in the spectrum.

Look at the spectrum for the orange solution. Do the wavelengths where the peaks occur in this spectrum match the positions of the peaks in the spectra for the red and yellow solutions? Do the absorbance values at the peaks in the spectrum for the orange solution match the absorbance values at the peaks in the spectra for the red and yellow solutions? What relationship (if any) exists between the absorption spectrum for the orange solution and the spectra for the red and yellow solutions? Consider the conclusions you drew above about the effect of dilution. Determine the ratio of volumes of the red and yellow stock solutions mixed together to prepare the orange solution; use your spectra and consider the conclusions you drew. Compare the ratio of volumes that you determined from the absorption spectra with the volume ratio calculated from the recorded volumes used to prepare the orange solution.

2-2 EXPERIMENT 2 ABSORPTION SPECTRUM

ABSORPTION SPECTRUM

REPORT FORM

Absorption Spectrum of Red Solution

Wavelength (nm) Absorbance Wavelength (nm) Absorbance 350 450 360 460 370 470 380 480 390 490 400 500 410 510 420 520 430 530 440 540

Wavelength(s) where the peak(s) occur(s)

2-3 ABSORPTION SPECTRUM EXPERIMENT 2

Absorption Spectrum of Yellow Solution

Wavelength (nm) Absorbance Wavelength (nm) Absorbance 350 450 360 460 370 470 380 480 390 490 400 500 410 510 420 520 430 530 440 540

Wavelength(s) where the peak(s) occur(s)

2-4 EXPERIMENT 2 ABSORPTION SPECTRUM

Absorption Spectrum of Diluted Stock Solution

Wavelength (nm) Absorbance Wavelength (nm) Absorbance 350 450 360 460 370 470 380 480 390 490 400 500 410 510 420 520 430 530 440 540

Color of stock solution used to prepare diluted solution

Wavelength(s) where the peak(s) occur(s)

2-5 ABSORPTION SPECTRUM EXPERIMENT 2

Absorption Spectrum of Orange Solution

Wavelength (nm) Absorbance Wavelength (nm) Absorbance 350 450 360 460 370 470 380 480 390 490 400 500 410 510 420 520 430 530 440 540

Volume of red solution used

Volume of yellow solution used

Wavelength(s) where the peak(s) occur(s)

2-6 EXPERIMENT 2 ABSORPTION SPECTRUM

Do the peaks for the stock colored solution and your diluted solution occur at the same wavelength(s)? If not, are the wavelengths similar? Are the absorbance values at the peaks for the two solutions the same?

What conclusions can you draw about the effect that diluting the stock solution has on the absorption spectrum for the solution? Try to be as specific as possible.

If you mixed equal volumes of the diluted stock solution and distilled water, predict the absorbance value you would expect at the peak in the spectrum.

Do the wavelengths where the peaks occur in the spectrum for the orange solution match the peak positions in the spectra for the red and yellow solutions? Do the absorbance values at the peaks in the spectrum for the orange solution match the absorbance values for the corresponding peaks in the spectra for the red and yellow solutions?

2-7 ABSORPTION SPECTRUM EXPERIMENT 2

What relationship (if any) exists between the absorption spectrum for the orange solution and the spectra for the red and yellow solutions? Consider the conclusion you drew about the effect of dilution.

Use information from the recorded spectra to determine the ratio of volumes of the red and yellow solutions mixed to prepare the orange solution.

Compare the volume ratio calculated using information from the spectra with the volume ratio calculated from the volumes used to prepare the orange solution.

2-8 EXPERIMENT 2 ABSORPTION SPECTRUM

NOTES TO INSTRUCTOR

The peaks occur at approximately 500nm and 420nm for the stock red solution and stock yellow solution, respectively. The diluted stock solution will have a peak at the same wavelength as the stock solution from which the diluted solution was prepared. If the dilution was done properly, the absorbance at the peak in the diluted stock solution is one-half the absorbance value at the peak in the stock solution. The students should conclude that dilution does not affect the peak position but does affect the absorbance value; they should also conclude that the amount of absorbing substance in the solution and absorbance are directly proportional. The predicted absorbance of the solution prepared by diluting the diluted stock solution with water is one-half of the absorbance seen with the diluted stock solution or one-quarter of the absorbance originally observed with the stock solution.

In preparing the orange solution, the red solution dilutes the yellow solution and the yellow solution dilutes the red solution. The absorption spectrum of the orange solution is a composite or sum of the spectra for the diluted stock solutions. At each wavelength in the absorption spectrum for the orange solution, the absorbance equals the sum of the absorbances at that wavelength of the diluted red solution and the diluted yellow solution. If the absorbance at the peak in the spectrum of the stock red solution is approximately the same value as the absorbance at the peak in the spectrum for the yellow solution, then an orange solution prepared by mixing equal volumes of the stock red and yellow solutions will have an absorption spectrum which has only one peak, at approximately 450nm. As the volume ratio of red solution to yellow solution increases above one-to-one, the position of the single peak in the spectrum shifts toward 500nm. As the volume ratio decreases below one-to-one, the position of the single peak shifts toward 420nm. A volume range of 3-7mL for each stock solution and a total volume of 10mL are suggested so that similar volumes of the two stock solutions will be combined; this insures that neither the numerator nor denominator in Equation 1 below will be a small number.

At any wavelength, let Ar = absorbance of stock red solution, Ay = absorbance of stock yellow solution, A0 = absorbance of orange solution, Vr = volume of stock red solution used to prepare the orange solution, and Vy = volume of stock yellow solution used to prepare the orange solution. At any wavelength, A0 has contributions from the red and yellow components present. If Beer's Law is obeyed, the volume ratio Vr/Vy is given by:

Vr Ao - Ay (1) = Vy Ar - A0

To calculate the volume ratio, use the absorbance of the red solution and the yellow solution at the wavelength where the peak occurs in the spectrum for the red solution or the yellow solution. Then, use the absorbance of the orange solution at the same wavelength. Selecting a wavelength where a peak occurs insures that one absorbance value is large.

2-9 ABSORPTION SPECTRUM EXPERIMENT 2

Each stock colored solution can be prepared by adding one drop of food color to 250mL of distilled water. You may wish to check the absorbance value of each stock colored solution you prepared. An absorbance value of 0.8-1.0 at the peak in the absorption spectrum is acceptable. You can add more food coloring to the stock solution and mix thoroughly until the desired absorbance value is achieved. An absorbance value of this magnitude insures that a substantial decrease in absorbance at the peak will be observed for the diluted stock solution.

Your students may find that it is easier to directly read percent transmittance, %T, from the spectrophotometer rather than absorbance, A. If so, %T can be read and A then calculated using Equation 2 below:

A = 2.000 - log (%T) (2)

Whether the students work individually or in groups, the number of solution spectra that an individual or group records are determined by the number of available spectrophotometers and the length of time allotted for lab work. If there is one spectrophotometer for every 1-2 students and a 2-3 hour lab time, the experiment could be done individually and each student would record the spectrum of each of the four solutions studied in the experiment. The experimental procedure was written for this possibility. If there are a sufficient number of spectrophotometers but only 1 hour available, the students can work in groups of 4, where each student records the spectrum for only one of the four solutions and the students later exchange their spectral data. If there is a limited number of spectrophotometers but a 2-3 hour time period is available, the students can work in groups of 2 or 3 and each group records the spectra of all four solutions. With a limited number of spectrophotometers and only one hour available, the students can work in groups of 2-3, each group records the spectrum of one solution, and groups later exchange data. If the spectrophotometers are old and have not been calibrated in a long time, the recorded position of each peak for a solution may vary slightly, depending on the spectrophotometer used. If this occurs, it will only be a complication where spectra are being exchanged among individuals or groups and all spectra for the four solutions were not recorded using the same spectrophotometer. Consequently, you may need to warn the students to not expect to see a perfect match in peak positions when comparing solutions containing the same colored component.

Materials needed, per group:

20mL aqueous solution of red food coloring 20mL aqueous solution of yellow food coloring 50mL distilled water 10mL graduated cylinder 2 50-mL beakers 2 cuvettes Spectrophotometer Box of disposable wipes Plastic disposable droppers

2-10 EXPERIMENT 3

DETERMINATION OF CONCENTRATION USING BEER'S LAW

INTRODUCTION

Visible light is that portion of the electromagnetic spectrum which has wavelengths in the range from approximately 380nm to 760nm. Different wavelengths of visible light appear as different colors to your eyes. All the wavelengths or colors of visible light, taken together, appear as white light; an absence of all wavelengths or colors appears black. If a beam of visible light is shone on a colored solution prepared by dissolving a colored solute in a colorless solvent, the colored component of the solution will typically absorb some of the wavelengths in the light beam and transmit other wavelengths. The color of the solution is determined by the wavelengths which are transmitted through the solution.

The approximate relation among wavelength absorbed, wavelength transmitted, and color observed can be illustrated using Figure 1.

Figure 1. A Diagram Showing the Relation Between Color and Wavelength Range.

The range of wavelengths between 380nm and 760nm is divided into six smaller ranges: 380nm - 430nm, 430nm - 480nm, etc. Each small range of wavelengths has the color, shown in the sector between the wavelengths, associated with it: violet with 380nm - 430nm, blue with 430nm - 480nm, etc. Complementary colors are approximately on opposite sides of the figure; green and red, yellow and violet, and orange and blue are complementary colors. If the solution transmits the wavelengths within one of the small ranges shown, and absorbs some or all of the wavelengths within the other ranges, the color of the solution seen will be the color associated with the wavelength range transmitted. If a solution absorbs wavelengths that are all within one of the ranges, and transmits the wavelengths in all of the other ranges, the color observed is often the color complement of the color associated with the wavelength range where absorption occurs. For example, if the solution absorbs wavelengths in the 480nm - 560nm range (the green color range), and transmits all other wavelength ranges, the solution color observed will be the complementary color of green; the solution will be red.

3-1 DETERMINATION OF CONCENTRATION USING BEER’S LAW EXPERIMENT 3

A spectrophotometer is an instrument which can be used to determine which wavelengths in the visible region are transmitted and which are absorbed by a colored solution. The instrument also can be used to determine the degree or extent of absorption at any wavelength. A plot of degree of absorption versus wavelength is called an absorption spectrum. The absorption spectrum is of interest since it is characteristic of the colored substance in solution and can therefore be used to identify the presence of that substance. At any wavelength, the degree of absorption is related to the concentration of the absorbing substance in the solution.

Let I0 represent the intensity of a single wavelength light beam striking a sample contained in a sample cell of a spectrophotometer, and let It represent the intensity of the light after the beam has passed through the sample. The percent transmittance of the sample, %T, is defined as

I (1) %T = —t x 100% Io

The value of It, and hence of %T, depends on the wavelength of the light. The corresponding degree of absorption is called the absorbance, A, of the solution at that wavelength. The relation between A and %T is:

100% (2) A = log (———) %T

(When both %T and A values appear on the same spectrophotometer scale, it can be seen that an absorbance value of zero equals 100% transmittance, so either A = 0 or %T = 100% refers to the same reference point.) The absorbance at a specified wavelength is a function of three factors: the path length, designated by b, which is the width of the sample through which the light passes (normally measured in cm); the concentration, designated by c, of the absorbing substance; and the absorptivity, designated by a, which represents the ability of the substance to absorb light at that specified wavelength. The relationship between A and the three factors is given by Beer's Law:

A = abc (3)

The concentration of the absorbing substance is usually expressed as a molar concentration. Molarity as the concentration unit will be assumed for the remainder of this experiment. When molarity is used, the absorptivity, a, is called the molar absorptivity and is often designated ,. If c has mol-L-1 as its unit, and b has cm as its unit, the unit for , is L-mol-1-cm-1, making A a unitless quantity. For a fixed path length and a specified wavelength, Beer's Law predicts that the absorbance of a solution is directly proportional to the molar concentration of the light-absorbing substance. (Remember, as the concentration of the absorbing substance becomes larger, a larger percentage of the light is absorbed by the sample and a smaller percentage of light is transmitted.) For a substance at fixed concentration and path length, absorbance changes as wavelength changes because the value for a ( or ,) depends on the wavelength of light.

Beer's Law can be used to determine the concentration of an absorbing substance in a solution. For greatest sensitivity in determining the concentration, the measurement of absorbance (or percent

3-2 EXPERIMENT 3 DETERMINATION OF CONCENTRATION USING BEER’S LAW

transmittance) is done at the wavelength where the maximum absorbance (or minimum %T) is observed for the substance. One way an unknown concentration can be determined is to first prepare a standard solution—a solution containing a known concentration of the absorbing species being studied—and determine the absorbance for that solution. The known value for c and the experimentally measured value for A can then be used to calculate ab in Equation 3. If a sample of the same absorbing substance, but of unknown concentration, is analyzed using the same wavelength and path length as the standard solution, ab for the standard solution and the unknown solution have the same value; the measured absorbance of the unknown solution, the value for ab, and Equation 3 can be used to calculate the concentration of the absorbing substance in the unknown. This method is somewhat risky, however. First, the method assumes that there have been no errors made in the preparation of the standard solution, because there is no additional independent information for comparison. In addition, the method assumes that the absorbing substance follows Beer's Law at all concentrations. In reality, deviations from Beer's Law can occur at high concentration of the absorbing substance.

A better way to determine an unknown concentration is to construct a standard curve; the curve is also called a calibration curve or Beer's Law plot. This curve can correct for deviations from Beer's Law. Typically, at least five standard solutions of different concentrations are prepared. Concentrations for the standard solutions are selected to span the expected concentration range of the samples to be analyzed. The absorbance value for each standard solution is determined (either directly measured or calculated from a measured %T) at the wavelength where maximum absorbance occurs. The absorbance values are plotted versus the corresponding molar concentrations of the absorbing substance in the standard solutions to create the standard curve. If there are no deviations from Beer's Law, the points will lie on or very close to the best straight line through the points. If there are deviations, there will be curvature in the line. The unknown concentration of the absorbing substance, in another solution, can then be determined by obtaining A (at the same wavelength and with the same path length as were used with the standard solutions) and using the standard curve to determine the concentration corresponding to A for the unknown solution.

Figure 2. A Representative Standard Curve.

3-3 DETERMINATION OF CONCENTRATION USING BEER’S LAW EXPERIMENT 3

An example of a standard curve is shown in Figure 2. If, for example, a solution of unknown molar concentration of the absorbing substance has an absorbance of 0.55, the concentration in that unknown can be determined by reading across (along the dotted line) to the best straight line, then reading down to determine the concentration value corresponding to A = 0.55; here, that concentration would be approximately 0.035 M.

This experiment will give you experience in utilizing Beer's Law to determine the concentration of an unknown. You will also gain experience in using the spectrophotometer and in preparing solutions of known concentration by dilution. You first measure %T values over a wavelength range in the visible region to determine the wavelength where the minimum %T (i.e., maximum A) occurs for the absorbing substance in a stock colored aqueous solution. All subsequent %T measurements are done only at the wavelength where the minimum %T is observed. Standard solutions will be prepared by diluting different volumes of the stock colored aqueous solution of the absorbing substance to the same total solution volume. The %T of each standard solution will be measured and the corresponding absorbance value calculated. A standard curve will be constructed by plotting absorbance versus molar concentration for the standard solutions. The best straight line through the points will give the standard curve. The %T of a solution of unknown concentration will be measured and the absorbance value of that solution calculated. Finally, the standard curve will be used to determine the molar concentration of the absorbing substance in the unknown.

EXPERIMENTAL PROCEDURE

Always wear safety goggles while performing the experiment.

Reagents and Equipment Needed

35 mL Stock Dye Solution 50-mL beaker 10-mL graduated cylinder 10-mL volumetric flask with stopper 4 15 x 125 mm test tubes Cuvette Spectrophotometer Plastic disposable droppers Unknown

Preparation of Solutions for Generating the Beer's Law Plot

The solution in the bottle labeled Stock Dye Solution is the stock colored aqueous solution to be used in this experiment. It was prepared by adding red food coloring to distilled water (most likely, the red food coloring consists of a single molecular substance dissolved in water). You will prepare the aqueous solutions used to generate the standard curve by diluting the stock solution with distilled water. The unknowns in this experiment were also prepared by diluting the stock solution with distilled water.

3-4 EXPERIMENT 3 DETERMINATION OF CONCENTRATION USING BEER’S LAW

Obtain 35 mL of the Stock Dye Solution in a clean, dry 5-mL beaker. Record the molar concentration of the Stock Dye Solution, which is given on the label of the bottle; this is the concentration of the absorbing substance. If you spill on yourself the Stock Dye Solution or any solution prepared from it, rinse the affected area with water.

Rinse a clean 10-mL graduated cylinder with 1-2 mL of the Stock Dye Solution; discard the rinsing. Use the graduated cylinder to transfer 2.0 mL of Stock Dye Solution to a clean 10-mL volumetric flask. Record the volume transferred to ±0.1 mL. Add enough distilled water to the 10-mL volumetric flask to fill the flask to the calibration mark. Invert to thoroughly mix the contents of the volumetric flask. Transfer the solution to a clean, dry 15 x 125 mm or larger test tube; label the test tube Solution 1. Rinse the volumetric flask with two portions of distilled water and discard the rinsings. Dispose of all liquid wastes in this experiment by pouring down the drain and flushing with water.

Prepare Solutions 2, 3, and 4 using 4.0 mL, 6.0 mL, and 8.0 mL, respectively, of Stock Dye Solution. Follow the procedure described for Solution 1; the 10-mL graduated cylinder used to measure the Stock Dye Solution does not need to be rinsed between solution preparations.

Spectroscopy

Rinse a clean cuvette with 1-2 mL of the Stock Dye Solution; discard the rinsing. Fill the cuvette about two-thirds full with Stock Dye Solution, and clean and dry the outside of the cuvette with a disposable wipe. Using a spectrophotometer, record the percent transmittance, %T, to ±0.1%, for the Stock Dye Solution from 400nm to 600nm at 20nm intervals. Use a second cuvette two-thirds filled with distilled water for the 100%T adjustment of the instrument at each wavelength. Remember to also set the 0%T adjustment at each wavelength, with the sample compartment empty and the sample compartment cover closed. Determine the wavelength where the minimum %T value (i.e. maximum absorbance value, A) is observed; you may want to check at 10nm intervals on either side of the wavelength where the smallest %T value is first seen to determine the wavelength of minimum %T more precisely. Record the wavelength where the minimum %T is observed. Discard the contents of the cuvette, rinse the cuvette with 1-2 mL of distilled water, and discard the rinsing.

Set the spectrophotometer at the wavelength where the minimum %T value was observed with the Stock Dye Solution. Perform a 0%T and l00%T adjustment of the spectrophotometer at this wavelength. Record %T, to ±0.1%, for Solutions 1-4 and the unknown. For each of these %T measurements, rinse the cuvette with 1-2 mL of the solution to be measured, fill the cuvette approximately two-thirds full with the solution, and record %T; start with Solution 1. After measuring %T, pour the solution from the cuvette back into its test tube. Solutions 1-4 can be discarded at the conclusion of the experiment and the test tubes rinsed with distilled water.

Obtain an unknown and record its number. Before measuring %T of the unknown, first rinse the cuvette with 1-2 mL of distilled water, discard that rinsing, then rinse with 1-2 mL of the unknown. After measuring the %T of the unknown, return the container with the remaining unknown to your lab instructor.

3-5 DETERMINATION OF CONCENTRATION USING BEER’S LAW EXPERIMENT 3

CALCULATIONS

Use the molar concentration of the absorbing substance in the Stock Dye Solution to calculate the molar concentration of the absorbing substance in Solutions 1-4, where measured volumes of Stock Dye Solution and distilled water were combined.

Calculate the absorbance for each of Solutions 1-4, the Stock Dye Solution, and the unknown from the %T values measured at the wavelength where the minimum %T value was observed with the Stock Dye Solution.

Generate the standard curve by plotting the absorbance of each numbered solution versus its calculated molar concentration of absorbing substance. Also plot absorbance versus molar concentration for the Stock Dye Solution. Draw the best straight line through the data points, including the origin as an additional point.

Use the standard curve and the absorbance for the unknown to determine the molar concentration of the absorbing substance in the unknown.

3-6 EXPERIMENT 3 DETERMINATION OF CONCENTRATION USING BEER’S LAW

DETERMINATION OF CONCENTRATION USING BEER'S LAW

REPORT FORM

Number of unknown

Molar concentration of Stock Dye Solution

Number of Solution for Volume of Stock Dye Solution Total Volume of Solution Generating Standard Curve Used Prepared 1 2 3 4

Absorption Spectrum of Stock Dye Solution

Wavelength %T Wavelength %T

Wavelength selected for measuring %T

3-7 DETERMINATION OF CONCENTRATION USING BEER’S LAW EXPERIMENT 3

Solution %T at Wavelength Selected Absorbance 1 2 3 4 Stock Dye Unknown

Solution Molar Concentration of Absorbing Substance 1 2 3 4

Calculations:

Molar concentration of absorbing substance in the unknown

3-8 EXPERIMENT 3 DETERMINATION OF CONCENTRATION USING BEER’S LAW

NOTES TO INSTRUCTOR

The standard photomultiplier tube in older models of the SPECTRONIC 20 operates over a wavelength range of 340nm - 600nm. The wavelength where the minimum %T (i.e. maximum A) occurs is approximately 500nm when red food coloring is used to prepare the Stock Dye Solution. Yellow food coloring could also be used to prepare the Stock Dye Solution; its minimum %T occurs at approximately 420nm. Green food coloring gives a solution showing two peaks in the absorption spectrum; the peak at approximately 620nm has a larger absorbance than the second peak at approximately 420nm. It is preferable, for sensitivity reasons, to be at the position of the largest peak; this excludes green food coloring as the color source. However, if sensitivity is not an important concern, green food coloring could be used to prepare the Stock Dye Solution, if measurements are then carried out at 420nm. Blue food coloring gives a solution with a minimum %T at approximately 620nm; blue food coloring cannot be used with the standard photomultiplier tube in the instrument. However, if the instrument has the wide range photomultiplier tube in it, which has a range of 400nm - 700nm, any of the four food colorings can be used to prepare the Stock Dye Solution. The newest models of the SPECTRONIC 20 have a solid state detector with a range of 340nm - 950nm; any of the four food colorings can be used to prepare the Stock Dye Solution.

If there are a sufficient number of 10-mL volumetric flasks available that each student can have four flasks, Solutions 1-4 can be left in the flasks in which they were prepared and do not need to be transferred to test tubes. If no 10-mL volumetric flasks are available, the students can use the following procedure: add the desired volume of Stock Dye Solution to the 10-mL graduated cylinder; add distilled water until the liquid level is at the 10-mL mark in the cylinder; transfer the liquid to a large test tube; thoroughly mix the solution with a clean glass stirring rod; pour several milliliters of the solution from the test tube back into the graduated cylinder and then pour the liquid from the cylinder back into the test tube (this insures all Stock Dye Solution was transferred to the test tube); mix again with the stirring rod; rinse the graduated cylinder with several milliliters of distilled water and discard the rinsing before beginning preparation of the next solution. Either a l0-mL graduated pipet or a 50-mL buret can be used instead of the 10-mL graduated cylinder to measure the volume of Stock Dye Solution used to prepare Solutions 1-4. The pipet or buret must be rinsed with the Stock Dye Solution.

A Stock Dye Solution having a %T of approximately 10% at the wavelength where the minimum %T value occurs is acceptable. This %T can be produced by adding approximately 20 drops of red food coloring to 1 L of distilled water. The actual molar concentration of the Stock Dye Solution is unimportant; simply fabricate a value for the label on the bottle of the Stock Dye Solution. The important operations in this experiment are performing the dilutions to prepare a set of standard solutions, carrying out the calculations of the concentrations, drawing the Beer's Law plot, and reading information from that plot.

3-9 DETERMINATION OF CONCENTRATION USING BEER’S LAW EXPERIMENT 3

For the unknown, provide 10 mL of solution in a glass container (vial or test tube) stoppered with a cork stopper. The cork stopper is needed only to prevent spillage or significant evaporation of water if the unknown is stored for a long period. To prepare an unknown, add 35 mL of Stock Dye Solution, measured using a 50-mL graduated cylinder, 50-mL buret, or 35-mL pipet, to a clean l00-mL volumetric flask; fill the flask with distilled water to the calibration mark and mix. This gives 10 unknowns of the same concentration. Other volumes of the Stock Dye Solution that could be used to prepare 100 mL of unknown are: 45 mL, 50 mL, 55 mL, and 65 mL. Any volume of Stock Dye Solution that will give an unknown with a concentration around the middle of the concentration range spanned by the Beer's Law plot and that is not one of the concentrations of the standard solutions prepared is acceptable. Alternatively, unknowns can be individually prepared if a unique concentration for each unknown is desired.

Amounts of reagents and equipment needed, per person:

35 mL Stock Dye Solution 50-mL beaker 10-mL graduated cylinder 10-mL volumetric flask with stopper 30 mL distilled water 4 15 x 125 mm test tubes Cuvette 10 mL unknown in cork-stoppered container 3 disposable plastic droppers for solution transfers

For the class as a whole:

Spectrophotometer(s) 1 cuvette filled with distilled water for each spectrophotometer 1 box disposable wipes for each spectrophotometer

3-10 EXPERIMENT 4

IMPORTANCE OF WAVELENGTH SELECTED IN THE BEER'S LAW EXPERIMENT

INTRODUCTION

Visible light is that portion of the electromagnetic spectrum which has wavelengths in the range from approximately 380nm to 760nm. Different wavelengths of visible light appear as different colors to your eyes. All the wavelengths or colors of visible light, taken together, appear as white light; an absence of all wavelengths or colors appears black. If a beam of visible light is shone on a colored solution prepared by dissolving a colored solute in a colorless solvent, the colored component of the solution will typically absorb some of the wavelengths in the light beam and transmit other wavelengths. The color of the solution is determined by the wavelengths which are transmitted through the solution.

The approximate relation among wavelength absorbed, wavelength transmitted, and color observed can be illustrated using Figure 1.

Figure 1. A Diagram Showing the Relation Between Color and Wavelength Range.

The range of wavelengths between 380nm and 760nm is divided into six smaller ranges: 380nm - 430nm, 430nm - 480nm, etc. Each small range of wavelengths has the color, shown in the sector between the wavelengths, associated with it: violet with 380nm - 430nm, blue with 430nm - 480nm, etc. Complementary colors are approximately on opposite sides of the figure; green and red, yellow and violet, and orange and blue are complementary colors. If the solution transmits the wavelengths within one of the small ranges shown, and absorbs some or all of the wavelengths within the other ranges, the color of the solution seen will be the color associated with the wavelength range transmitted. If a solution absorbs wavelengths that are all within one of the ranges, and transmits the wavelengths in all of the other ranges, the color observed is often the color complement of the color associated with the wavelength range where absorption occurs. For example, if the solution absorbs wavelengths in the 480nm - 560nm

4-1 IMPORTANCE OF WAVELENGTH SELECTED IN THE BEER’S LAW EXPERIMENT EXPERIMENT 4

range (the green color range), and transmits all other wavelength ranges, the solution color observed will be the complementary color of green; therefore, the solution will be red.

A spectrophotometer is an instrument which can be used to determine which wavelengths in the visible region are transmitted and which are absorbed by a colored solution. The instrument also can be used to determine the degree or extent of absorption at any wavelength. A plot of degree of absorption versus wavelength is called an absorption spectrum. The absorption spectrum is of interest since it is characteristic of the colored substance in solution and can therefore be used to identify the presence of that substance. At any wavelength, the degree of absorption is related to the concentration of the absorbing substance in the solution.

Let IO represent the intensity of a single wavelength light beam striking a sample contained in a sample cell of a spectrophotometer, and let It represent the intensity of the light after the beam has passed through the sample. The percent transmittance of the sample, %T, is defined as

I (1) %T = —t x 100% Io

The value of It, and hence of %T, depends on the wavelength of the light. The corresponding degree of absorption is called the absorbance, A, of the solution at that wavelength. The relation between A and %T is:

100% (2) A = log (———) %T

(When both %T and A values appear on the same spectrophotometer scale, it can be seen that an absorbance value of zero equals 100% transmittance, so either A = 0 or %T = 100% refers to the same reference point.) The absorbance at a specified wavelength is a function of three factors: the path length, designated by b, which is the width of the sample through which the light passes (normally measured in cm); the concentration, designated by c, of the absorbing substance; and the absorptivity, designated by a, which represents the ability of the substance to absorb light at that specified wavelength. The relationship between A and the three factors is given by Beer's Law:

A = abc (3)

The concentration of the absorbing substance is usually expressed as a molar concentration. Molarity as the concentration unit will be assumed for the remainder of this experiment. When molarity is used, the absorptivity, a, is called the molar absorptivity and is often designated ,. If c has mol-L-1 as its unit, and b has cm as its unit, the unit for , is L-mol-1-cm-1, making A a unitless quantity. For a fixed path length and a specified wavelength, Beer's Law predicts that the absorbance of a solution is directly proportional to the molar concentration of the light-absorbing substance. (Remember, as the concentration of the absorbing substance becomes larger, a larger percentage of the light is absorbed by the sample and a smaller percentage of light is transmitted.) For a substance at fixed concentration and path length, absorbance changes as wavelength changes because the value for a (or ,) depends on the wavelength of light.

4-2 EXPERIMENT 4 IMPORTANCE OF WAVELENGTH SELECTED IN THE BEER’S LAW EXPERIMENT

Beer's Law is used to determine the concentration of an absorbing substance in a solution. One way an unknown concentration can be determined is to first prepare a standard solution—a solution containing a known concentration of the absorbing species being studied—and determine the absorbance for that solution. The known value for c and the experimentally measured value for A can then be used to calculate ab in Equation 3. If a sample of the same absorbing substance, but of unknown concentration, is analyzed using the same wavelength and path length as the standard solution, ab for the standard solution and the unknown solution have the same value; the measured absorbance of the unknown solution, the value for ab, and Equation 3 can be used to calculate the concentration of the absorbing substance in the unknown. This method is somewhat risky, however. First, the method assumes that there have been no errors made in the preparation of the standard solution, because there is no additional independent information for comparison. In addition, the method assumes that the absorbing substance follows Beer's Law at all concentrations. In reality, deviations from Beer's Law can occur at high concentration of the absorbing substance.

A better way to determine an unknown concentration is to construct a standard curve; the curve is also called a calibration curve or Beer's Law plot. This curve can correct for deviations from Beer's Law. Typically, at least five standard solutions of different concentrations are prepared. Concentrations for the standard solutions are selected to span the expected concentration range of the samples to be analyzed. The absorbance value for each standard solution is determined (either directly measured or calculated from a measured %T). The absorbance values are plotted versus the corresponding molar concentrations of the absorbing substance in the standard solutions to create the standard curve. If there are no deviations from Beer's Law, the points will lie on or very close to the best straight line through the points. If there are deviations, there will be curvature in the line. The unknown concentration of the absorbing substance, in another solution, can then be determined by obtaining A (at the same wavelength and with the same path length as were used with the standard solutions) and using the standard curve to determine the concentration corresponding to A for the unknown solution.

Figure 2. A Representative Standard Curve.

4-3 IMPORTANCE OF WAVELENGTH SELECTED IN THE BEER’S LAW EXPERIMENT EXPERIMENT 4

An example of a standard curve is shown in Figure 2. If, for example, a solution of unknown molar concentration of the absorbing substance has an absorbance of 0.55, the concentration in that unknown can be determined by reading across (along the dotted line) to the best straight line, then reading down to determine the concentration value corresponding to A = 0.55; here, that concentration would be approximately 0.035 M.

In either of the two procedures described above for determining an unknown concentration of an absorbing substance in a solution, the measurement of absorbance (or percent transmittance) is done at the wavelength where the maximum absorbance (or minimum %T) is observed for the substance. This experiment investigates why this wavelength is used in the quantitative determination of concentration. You will first measure %T values for the absorbing substance in a stock colored aqueous solution over a wavelength range in the visible region in this experiment. Three wavelengths will then be selected: the wavelength at the top of the largest absorption peak in the absorption spectrum, where the minimum %T (maximum A) occurs; a wavelength along the baseline in the spectrum, where the maximum %T (minimum A) occurs; and a wavelength on the sloping side of the largest absorption peak in the spectrum, where the absorbance is intermediate between the maximum and minimum A values. All subsequent %T measurements are done at each of the three wavelengths selected.

Standard solutions will be prepared by diluting different volumes of the stock colored aqueous solution of the absorbing substance to the same total solution volume. The %T of each standard solution will be measured and the corresponding absorbance value calculated. A standard curve for each wavelength selected will be constructed by plotting absorbance versus molar concentration for the standard solutions at that wavelength. The best straight line through the points will give the standard curve. The %T of a solution of known concentration will be measured and the absorbance value of that solution calculated. Finally, the relevant standard curve will be used to determine the molar concentration of the absorbing substance in the known. For each wavelength you will compare the concentration determined from the appropriate standard curve with the known concentration. From this, you will decide if it is necessary to do absorbance measurements at the wavelength of maximum absorbance.

EXPERIMENTAL PROCEDURE

Always wear safety goggles while performing the experiment.

Reagents and Equipment Needed

40 mL Stock Dye Solution 100-mL beaker 10-mL graduated cylinder 10-mL volumetric flask with stopper 4 15 x 125 mm test tubes Cuvette Spectrophotometer Plastic disposable droppers

4-4 EXPERIMENT 4 IMPORTANCE OF WAVELENGTH SELECTED IN THE BEER’S LAW EXPERIMENT

Preparation of Solutions

The solution in the bottle labeled Stock Dye Solution is the stock colored aqueous solution to be used in this experiment. It was prepared by adding red food coloring to distilled water (most likely, the red food coloring consists of a single molecular substance dissolved in water). You will prepare the aqueous solutions used to generate the standard curve by diluting the stock solution with distilled water. The known in this experiment is also prepared by diluting the stock solution with distilled water.

Obtain 40 mL of the Stock Dye Solution in a clean, dry 100-mL beaker. Record the molar concentration of the Stock Dye Solution, which is given on the label of the bottle; this is the concentration of the absorbing substance. If you spill on yourself the Stock Dye Solution or any solution prepared from it, rinse the affected area with water.

Rinse a clean 10-mL graduated cylinder with 1-2 mL of the Stock Dye Solution; discard the rinsing. Use the graduated cylinder to transfer 2.0 mL of Stock Dye Solution to a clean 10-mL volumetric flask. Record the volume transferred to ±0.1 mL. Add enough distilled water to the 10-mL volumetric flask to fill the flask to the calibration mark. Invert to thoroughly mix the contents of the volumetric flask. Transfer the solution to a clean, dry 15 x 125 mm or larger test tube; label the test tube Solution 1. Rinse the volumetric flask with two portions of distilled water and discard the rinsings. Dispose of all liquid wastes in this experiment by pouring down the drain and flushing with water.

Prepare Solutions 2, 3, and 4 using 4.0 mL, 6.0 mL, and 8.0 mL, respectively, of Stock Dye Solution. Follow the procedure described for Solution 1; the 10-mL graduated cylinder used to measure the Stock Dye Solution does not need to be rinsed between solution preparations.

Select a volume of the Stock Dye Solution that will give a solution with a molar concentration of the absorbing substance around the middle of the concentration range spanned by the standard curve and that is not one of the concentrations of the standard solutions prepared. Use the 10-mL graduated cylinder to transfer the selected volume of Stock Dye Solution to the 10-mL volumetric flask; record the volume transferred to ±0.1 mL. Add enough distilled water to the 10-mL volumetric flask to fill the flask to the calibration mark, and invert to thoroughly mix the contents. The solution can be left in the volumetric flask and the flask labeled Known.

Spectroscopy

Rinse a clean cuvette with 1-2 mL of the Stock Dye Solution; discard the rinsing. Fill the cuvette about two-thirds full with Stock Dye Solution, and clean and dry the outside of the cuvette with a disposable wipe. Using a spectrophotometer, record the percent transmittance, %T, to ±0.1%, for the Stock Dye Solution from 400nm to 600nm at 20nm intervals. Use a second cuvette two-thirds filled with distilled water for the 100%T adjustment of the instrument at each wavelength. Remember to also set the 0%T adjustment at each wavelength, with nothing in the sample compartment and the sample compartment cover closed. Determine the wavelength where the minimum %T value (i.e. maximum absorbance value, A) is observed; you may want to check at 10nm intervals on either side of the wavelength where the smallest %T value is first seen to determine the wavelength of minimum %T more precisely. Select the wavelength where the maximum %T value (i.e. minimum A) is observed and select a wavelength where %T value is intermediate between the minimum and maximum %T values. Record the three wavelengths

4-5 IMPORTANCE OF WAVELENGTH SELECTED IN THE BEER’S LAW EXPERIMENT EXPERIMENT 4

selected. Discard the contents of the cuvette, rinse the cuvette with 1-2 mL of distilled water, and discard the rinsing.

Rinse the cuvette with 1-2 mL of Solution 1 and discard the rinsing. Fill the cuvette about two-thirds full with Solution 1, and clean and dry the outside of the cuvette with a disposable wipe. Record %T, to ±0.1%, at each of the three wavelengths selected. Remember to perform the 0%T and 100%T adjustments of the spectrophotometer at each wavelength. After measuring %T at the three wavelengths, pour the solution from the cuvette back into its test tube. Repeat the above procedure using Solutions 2-4 and the known. Before making the %T measurements using the known, first rinse the cuvette with 1-2 mL of distilled water, discard that rinsing, then rinse with 1-2 mL of the known. After making the measurements with the known, it can be poured back into the volumetric flask. Solutions 1-4 and the known can be discarded at the conclusion of the experiment and the test tubes and volumetric flask rinsed with distilled water.

CALCULATIONS

Use the molar concentration of the absorbing substance in the Stock Dye Solution to calculate the molar concentration of the absorbing substance in Solutions 1-4 and the known, where measured volumes of Stock Dye Solution and distilled water were combined.

At each of the three wavelengths you selected, calculate the absorbance for each of Solutions 1-4, the Stock Dye Solution, and the known from the measured %T values.

For each wavelength, generate the standard curve by plotting absorbance at that wavelength of each numbered solution versus its calculated molar concentration of absorbing substance. Also plot absorbance versus molar concentration for the Stock Dye Solution. Draw the best straight line through the data points, including the origin as an additional point.

For each of the three wavelengths selected, use the standard curve and the absorbance for the known at that wavelength to determine the molar concentration of the absorbing substance in the known. Compare each concentration determined from a standard curve with the calculated concentration of the known. Were you able to determine the concentration of the known equally well at the three wavelengths you selected? Was the determination better at one wavelength than at the other two? Is it necessary to make %T measurements at the wavelength of minimum %T to obtain a good determination of the known's concentration? Is it desirable to make measurements at the wavelength of minimum %T? Use your experimental results to justify your conclusions.

4-6 EXPERIMENT 4 IMPORTANCE OF WAVELENGTH SELECTED IN THE BEER’S LAW EXPERIMENT

IMPORTANCE OF WAVELENGTH SELECTED IN THE BEER'S LAW EXPERIMENT

REPORT FORM

Molar concentration of Stock Dye Solution

Number of Solution Volume of Stock Total Volume of Molar Concentration for Generating Dye Solution Solution of Absorbing Standard Curve Used Prepared Substance 1 2 3 4 Known

Calculations:

4-7 IMPORTANCE OF WAVELENGTH SELECTED IN THE BEER’S LAW EXPERIMENT EXPERIMENT 4

Absorption Spectrum of Stock Dye Solution

Wavelength %T Wavelength %T

Wavelength of minimum %T selected

Wavelength of maximum %T selected

Wavelength of intermediate %T selected

%T at Wavelength Selected

Solution Minimum %T Intermediate %T Maximum %T 1 2 3 4 Stock Dye Known

4-8 EXPERIMENT 4 IMPORTANCE OF WAVELENGTH SELECTED IN THE BEER’S LAW EXPERIMENT

Calculated Absorbance at Wavelength Selected

Solution Minimum %T Intermediate %T Maximum %T 1 2 3 4 Stock Dye Known

Molar Concentration of Absorbing Substance from Calibration Curve at:

Wavelength of minimum %T

Wavelength of maximum %T

Wavelength of intermediate %T

Comparison of each molar concentration determined from a calibration curve with the calculated molar concentration of the known.

Were you able to determine the concentration of the known equally well at the three wavelengths you selected? Was the determination better at one wavelength than at the other two? Is it necessary to obtain a good determination of the known's concentration? Is it desirable to make measurements at the wavelength of minimum %T? Use your experimental results to justify your conclusions.

4-9 IMPORTANCE OF WAVELENGTH SELECTED IN THE BEER’S LAW EXPERIMENT EXPERIMENT 4

NOTES TO INSTRUCTOR

The standard photomultiplier tube in older models of the SPECTRONIC 20 operates over a wavelength range of 340nm - 600nm. The wavelength where the minimum %T (i.e. maximum A) occurs is approximately 500nm when red food coloring is used to prepare the Stock Dye Solution. Yellow food coloring could also be used to prepare the Stock Dye Solution; its minimum %T occurs at approximately 420nm. Green food coloring gives a solution showing two peaks in the absorption spectrum; the peak at approximately 620nm has a larger absorbance than the second peak at approximately 420nm. It is preferable, for sensitivity reasons, to be at the position of the largest peak; this excludes green food coloring as the color source. However, if sensitivity is not an important concern, green food coloring could be used to prepare the Stock Dye Solution, if measurements are then carried out at 420nm. Blue food coloring gives a solution with a minimum %T at approximately 620nm; blue food coloring cannot be used with the standard photomultiplier tube in the instrument. However, if the instrument has the wide range photomultiplier tube in it, which has a range of 400nm - 700nm, any of the four food colorings can be used to prepare the Stock Dye Solution. The newest models of the SPECTRONIC 20 have a solid state detector with a range of 340nm - 950nm; any of the four food colorings can be used to prepare the Stock Dye Solution.

If there are a sufficient number of 10-mL volumetric flasks available that each student can have five flasks, Solutions 1-4 can be left in the flasks in which they were prepared and do not need to be transferred to test tubes. If no 10-mL volumetric flasks are available, the students can use the following procedure: add the desired volume of Stock Dye Solution to the 10-mL graduated cylinder; add distilled water until the liquid level is at the 10-mL mark in the cylinder; transfer the liquid to a large test tube; thoroughly mix the solution with a clean glass stirring rod; pour several milliliters of the solution from the test tube back into the graduated cylinder and then pour the liquid from the cylinder back into the test tube (this insures all Stock Dye Solution was transferred to the test tube); mix again with the stirring rod; rinse the graduated cylinder with several milliliters of distilled water and discard the rinsing before beginning preparation of the next solution. Either a l0-mL graduated pipet or a 50-mL buret can be used instead of the 10-mL graduated cylinder to measure the volume of Stock Dye Solution used to prepare Solutions 1-4 and the known. The pipet or buret must be rinsed with the Stock Dye Solution.

A Stock Dye Solution having a %T of approximately 10% at the wavelength where the minimum %T value occurs is acceptable. This %T can be produced by adding approximately 20 drops of red food coloring to 1 L of distilled water. The actual molar concentration of the Stock Dye Solution is unimportant; simply fabricate a value for the label on the bottle of the Stock Dye Solution. Any volume of Stock Dye Solution between 3.5 mL and 6.5 mL is acceptable for preparing the known.

If Stock Dye Solution has a minimum %T of approximately 10%, any wavelength where the Stock Dye Solution shows a large %T ($ 80%) is acceptable to use as the wavelength where the maximum %T value occurs. The wavelength where the intermediate %T value occurs can be selected from either side of the absorption peak in the spectrum. Any wavelength where the Stock Dye Solution shows a %T value approximately half way between the minimum and maximum %T values observed is acceptable.

4-10 EXPERIMENT 4 IMPORTANCE OF WAVELENGTH SELECTED IN THE BEER’S LAW EXPERIMENT

Conclusions drawn which are supported by the student's experimental observations are most important in this experiment. Also important are the operations of performing the dilutions to prepare a set of standard solutions, carrying out the calculations of the concentrations, drawing the Beer's Law plot, and reading information from that plot. Percent difference can be calculated to compare the concentration of the known experimentally determined from a calibration curve with the concentration calculated from the volume of Stock Dye Solution diluted.

The best agreement (i.e. smallest percent difference) between experimental and calculated concentrations for the known is found using the data collected at the wavelength where the minimum %T value is observed with the Stock Dye Solution. The largest difference between A values for Solution 1 and the Stock Dye Solution (the least and most concentrated standard solutions, respectively) occurs at this wavelength. With a large spread in A values for the standard solutions, a concentration value found using the calibration curve is affected only slightly by a small error in the experimentally determined A value for that solution. Good agreement between experimental and calculated concentrations for the known may also occur at the wavelength where the intermediate %T value is observed with the Stock Dye Solution. The spread in A values for the standard solutions is less at this wavelength than at the wavelength of minimum %T value, however. It is extremely difficult to determine the concentration at the wavelength of maximum %T value. There is very little difference among the very small A values at this wavelength for the standard solutions.

It is not absolutely necessary to measure %T values at the wavelength where the maximum in the strongest absorption peak occurs but it is desirable. Small errors in the A values for individual standard solutions are less important with a larger spread of A values. Consequently, data in a Beer's Law experiment are usually collected at this wavelength. If the three standard curves in this experiment are plotted on the same graph with the same scale, the differences in the extent to which the A values for the standard solutions change at the three wavelengths used are illustrated.

Amounts of reagents and equipment needed, per person:

40 mL Stock Dye Solution 10-mL beaker 10-mL graduated cylinder 10-mL volumetric flask with stopper 40 mL distilled water 4 15 x 125 mm test tubes Cuvette 3 Disposable plastic droppers for transferring liquids

For the class as a whole:

Spectrophotometer(s) One cuvette filled with distilled water for each spectrophotometer One box disposable wipes for each spectrophotometer

4-11 IMPORTANCE OF WAVELENGTH SELECTED IN THE BEER’S LAW EXPERIMENT EXPERIMENT 4

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4-12 EXPERIMENT 5

RED CABBAGE pH INDICATOR

INTRODUCTION

An acid-base indicator is a compound whose color in aqueous solution depends on the solution pH. In this experiment, you will first use water to extract an acid-base indicator from red cabbage. The solution colors resulting from adding the aqueous solution of the extracted indicator to aqueous solutions of some common household products will be observed. The absorption spectrum for each indicator-household product combination will be obtained and the wavelength determined where the peak occurs in the spectrum. The pH of each household product solution will be measured. You will plot wavelength where the peak occurs versus solution pH to see if there is a correlation between solution color and pH.

EXPERIMENTAL PROCEDURE

Always wear safety goggles while performing the experiment

Reagents and Equipment Needed

Red cabbage Knife Blender 500-mL filter flask 9-cm Buchner funnel 9-cm Whatman No. 1 filter paper Rubber adapter 50-mL Erlenmeyer flask plus cork stopper 10 mL aqueous table salt solution 10 mL aqueous baking soda solution 10 mL aqueous Liquid Drano solution 10 mL aqueous toilet bowl cleaner solution 10 mL club soda 10 mL white vinegar 10-mL graduated cylinder 8 15 x 125 mm test tubes (or larger size) Vial of pH paper Plastic disposable droppers Glass stirring rod 2 Spectrophotometer cells Spectrophotometer

5-1 RED CABBAGE pH INDICATOR EXPERIMENT 5

Cut a handful of red cabbage into small pieces on a clean hard surface, and place the pieces in a blender half-filled with distilled water. Blend the mixture. Separate the liquid and solid portions by vacuum filtration using the 500-mL filter flask, 9-cm filter paper and Buchner funnel, and rubber adapter. Transfer some of the filtrate (10-20 mL is acceptable) to a 50-mL Erlenmeyer flask, and stopper the flask with a cork stopper; the remaining filtrate can be left in the filter flask, while the solid collected in the Buchner funnel can be discarded in the trash. Record the color of the indicator solution.

Using a clean 10-mL graduated cylinder, transfer 10 mL of distilled water to a clean, dry 15 x 125 mm (or larger) test tube. Use the depth of liquid in this test tube as a measure for 10 mL, and transfer 10 mL of each of the following liquids to separate clean, dry 15 x 125 mm (or larger) test tubes: tap water, white vinegar, aqueous table salt solution, aqueous baking soda solution, aqueous Liquid Drano solution, aqueous toilet bowl cleaner solution, and club soda. Use pH paper to determine the pH of distilled water, tap water, and each of the six aqueous household product solutions; record your pH observations. Be careful with each of the household product solutions, especially with the aqueous Liquid Drano and toilet bowl cleaner solutions. If you spill any of these solutions on yourself, immediately rinse the affected area with a large volume of tap water. Do not combine household product solutions.

Add 10-15 drops of the indicator solution (taken from the 50-mL beaker) to the 10-mL sample of distilled water; mix the contents of the test tube with a clean glass stirring rod, and record the color of the resulting solution. Repeat the procedure (add the same number of drops of indicator solution as was added to distilled water) and record the resultant solution color using the 10-mL samples of tap water and the six household product solutions; remember to thoroughly rinse the stirring rod between mixings.

Obtain two clean spectrophotometer cells. Rinse one cell with several milliliters of distilled water, discard the rinsing in a sink, and fill the cell two-thirds full with distilled water. This cell will have only distilled water in it for the duration of the experiment.

Rinse the second cell with several milliliters of the distilled water sample containing added indicator solution. Discard the rinsing in a sink and fill the cell two-thirds full with the distilled water containing indicator solution. Follow your instructor's directions for the operation of your spectrophotometer. Record the absorbance of the solution from 360nm to 600nm at 20nm intervals. Determine the wavelength where the maximum absorbance value is observed; you may want to check at 10nm intervals on either side of the wavelength where the largest absorbance value is first seen to determine the wavelength of maximum absorbance more precisely. Record the wavelength where the maximum absorbance is observed. Discard the contents of the cell in a sink, rinse the cell with several milliliters of distilled water, and discard the rinsing in a sink.

Repeat the procedure described above, substituting the tap water and six household product solutions with added indicator for the distilled water with indicator. For each of the household product solutions, after discarding the several milliliters of rinsing, and later the spectrophotometry sample in the cell, into a sink, flush the sink with a large volume of tap water.

At the conclusion of the experiment, discard the distilled water in the second cell and return the cells. Any liquid remaining in any test tube can be poured down a drain, followed by a large volume of tap water before the next solution is poured down the drain; the test tube can be rinsed with distilled water and the rinsing discarded in a sink. Any indicator solution remaining in the 50-mL Erlenmeyer flask or the 500-mL filter flask can be discarded in a sink and the glassware rinsed with distilled water.

5-2 EXPERIMENT 5 RED CABBAGE pH INDICATOR

CALCULATIONS

For each of the eight liquids to which the indicator was added, plot absorbance (along the y-axis) versus wavelength (along the x-axis), and draw a smooth curve through the points for a solution. Plot all absorption spectra on the same piece of graph paper or in a computer graphing program. Indicate the liquid to which each spectrum applies. What conclusions, if any, can you draw from examining the eight spectra? What is the purpose of including the solution prepared by combining indicator and distilled water?

Plot wavelength where the maximum absorbance value was observed versus measured pH for each of the eight liquids. Is there a linear relationship between the wavelength of maximum absorbance and pH? Is there any correlation between wavelength and pH here? If so, what?

5-3 RED CABBAGE pH INDICATOR EXPERIMENT 5

RED CABBAGE pH INDICATOR

REPORT FORM

Color of indicator solution

Liquid pH Color after Wavelength of Adding Indicator Maximum Absorbance

What is the purpose of including the solution prepared by combining indicator and distilled water?

5-4 EXPERIMENT 5 RED CABBAGE pH INDICATOR

What conclusions, if any, can you draw from examining the eight spectra?

Is there a linear relationship between the wavelength of maximum absorbance and pH?

Is there any correlation between wavelength and pH here? If so, what?

5-5 RED CABBAGE pH INDICATOR EXPERIMENT 5

Solution Absorbance

Wavelength (nm)

5-6 EXPERIMENT 5 RED CABBAGE pH INDICATOR

Solution Absorbance

Wavelength (nm)

5-7 RED CABBAGE pH INDICATOR EXPERIMENT 5

NOTES TO INSTRUCTOR

A kitchen blender can be used in preparing the indicator solution. Blend until the cabbage is a mush. A handful of cabbage makes enough indicator solution for an entire lab section. You could prepare the indicator solution before lab begins, or you could have the students work as a single group at the start of lab to prepare the solution. Since the indicator solution has a noticeable odor, the small volume of indicator solution is kept in a stoppered container and the filter flask containing the remaining indicator solution should be stored in a hood. The indicator solution does not keep well and should be prepared fresh each day it is to be used in lab.

Vinegar is approximately a 0.8 M aqueous solution of acetic acid; the white vinegar can be used “as is” from the bottle. Use white vinegar so that the color observed on adding the indicator solution comes only

from the indicator. To prepare 100 mL of aqueous baking soda solution that is 1.0 M NaHCO3, add 8.4 g of baking soda (NaHCO3) to a clean 100-mL volumetric flask, add some distilled water to the flask to dissolve the solid, fill the flask to the calibration mark with distilled water, and invert the flask several times to thoroughly mix the solution. The aqueous baking soda solution is basic; transfer the solution to a capped plastic bottle after preparing it. To prepare 100 mL of aqueous Liquid Drano solution, add 20 mL of Liquid Drano to 80 mL of distilled water, each measured with a 100-mL graduated cylinder, in a clean 250-mL Erlenmeyer flask; stopper with a cork stopper and swirl to mix. Transfer the basic solution into a capped plastic bottle. Use Vanish (or any other solid toilet bowl cleaner) to prepare the aqueous

toilet bowl cleaner solution; Vanish is 62% NaHSO4 by mass. To prepare 100 mL of an aqueous toilet bowl cleaner solution that is approximately 0.25 M NaHSO4, use a 100-mL graduated cylinder to transfer 100 mL of distilled water to a clean 500-mL Erlenmeyer flask; add 4.8 g of Vanish to the Erlenmeyer flask; after the foaming has subsided, swirl to dissolve as much of the remaining solid as possible; stopper the flask and allow the solution to stand overnight; decant the solution from the remaining undissolved solid into a plastic bottle and cap, and discard any solid remaining in the flask down the drain using a large volume of tap water. Table salt (NaCl) or iodized table salt can be used to prepare the aqueous table salt solution. To prepare 100 mL of an aqueous table salt solution that is 1.0 M NaCl, add 5.8 g of table salt to a clean 100-mL volumetric flask, add some distilled water to the flask to dissolve the solid, fill the flask to the calibration mark with distilled water, and invert the flask several times to thoroughly mix the solution. Club soda was selected as the carbonated beverage to study because it is colorless; it can be used “as is” from the bottle.

The indicator solution is purple. Addition of the indicator solution to distilled water dilutes the indicator solution and gives a pale purple solution. Addition of the indicator solution to the aqueous toilet bowl cleaner solution, white vinegar, or club soda gives a pale pink color. Aqueous table salt solution plus indicator solution gives a pale purple color. Aqueous baking soda solution plus indicator gives a pale aqua solution, and aqueous Liquid Drano solution with added indicator solution gives a pale yellow- green color. The combination of indicator solution and distilled water is done to determine the solution color that the indicator gives when there is no solute (other than dissolved carbon dioxide) present in the solvent, distilled water.

Each spectrum shows an increase in absorbance as the wavelength decreases from 400nm to 360nm. In each spectrum, this suggests that there would be a peak at a wavelength shorter than 360nm; it cannot be determined if this peak would occur at a similar, or identical, position in each spectrum. Spectra for the acidic solutions having a pH between approximately 1 and 5 show a peak at approximately 520nm. As the solution pH increases above 5, the peak shifts to a longer wavelength; this shift in peak position suggests that the solution color should change as solution pH increases above 5, as is observed. The peak

5-8 EXPERIMENT 5 RED CABBAGE pH INDICATOR has shifted enough to longer wavelengths that a peak no longer occurs between 500nm and 600nm in the spectrum for the strongly basic aqueous Liquid Drano solution. There is no linear relationship between peak position and pH.

If none of the eight samples is deleted, there is a substantial number of spectra to be recorded in this experiment. If the number of spectrophotometers is limited and/or there is only one hour of lab time available, have each student, or group of 2-3 students, add the indicator solution to one of the eight samples and record the solution's spectrum. Make sure that each of the solutions you want to have studied is selected by at least one student or group. The students can later exchange their observations, including the spectral data, they obtained.

Your students may find that it is easier to directly read percent transmittance, %T, from the spectrophotometer rather than absorbance, A. If so, %T can be read and A then calculated using

A = 2.000 - log (%T) (1)

5-9 RED CABBAGE pH INDICATOR EXPERIMENT 5

Materials needed, per lab section, to prepare the indicator solution:

Red cabbage Knife Blender 500-mL filter flask 9-cm Buchner funnel 9-cm Whatman No. 1 filter paper 50-mL Erlenmeyer flask plus cork stopper Rubber adapter Distilled water Plastic disposable dropper

Materials needed for spectroscopic study:

8 15 x 125 mm test tubes (or larger size) 10 mL club soda 10 mL white vinegar 10 mL aqueous baking soda solution 10 mL aqueous table salt solution 10 mL aqueous Liquid Drano solution 10 mL aqueous toilet bowl cleaner solution Vial of pH paper Glass stirring rod Plastic disposable dropper 2 spectrophotometer cells Spectrophotometer Box of disposable wipes

5-10 EXPERIMENT 6

SPECTROPHOTOMETRIC DETERMINATION OF NITRATE ION CONCENTRATION

INTRODUCTION

The nitrate ion concentration in an aquarium should be determined weekly. A concentration above 40 parts per million (ppm) can lead to microalgae blooms in fresh water and marine aquariums, and excessive nitrate ion levels can damage some aquatic plants. (A concentration of 1 ppm means 1 g of the solute of interest per 106 g of solution containing that solute.) A home testing kit to determine a nitrate ion concentration up to 140 ppm can be purchased at a pet store. To test, a sample of water is combined with two tablets from the test kit, and the color which develops in the solution is compared with a chart (supplied in the test kit) correlating color and concentration.

Development of a color on combining the water sample and testing reagents and change in color with change in concentration suggest that it might be possible to determine the nitrate ion concentration spectrophotometrically, using the reagents from the home test kit to create a colored solution. The feasibility of such a spectrophotometric method to determine the nitrate ion concentration will be studied in this experiment. You will first use the reagents and procedure from the home test kit with several standard solutions of known nitrate ion concentration to produce colored solutions. The absorption spectrum of each solution will be recorded to determine the wavelength for making absorbance measurements in a spectrophotometric procedure. The absorbance values at the wavelength selected will then be plotted versus the known concentrations of nitrate ion. A spectrophotometric method is feasible only if all of the following criteria are met: a peak (i.e., a maximum in the absorbance value) in the absorption spectrum (the plot of absorbance versus wavelength) occurs at the same wavelength in the spectrum for each standard solution studied; and, at the wavelength selected, the absorbance value is substantial for each standard solution and there is a linear relationship between absorbance and concentration shown by the calibration curve (the plot of absorbance at one wavelength versus concentration).

6-1 SPECTROPHOTOMETRIC DETERMINATION OF NITRATE ION CONCENTRATION EXPERIMENT 6

EXPERIMENTAL PROCEDURE

Always wear safety goggles while performing the experiment.

Reagents and Equipment Needed

5 13 x 100 mm test tubes (or larger size) 4 50-mL Erlenmeyer flasks 4 cork stoppers to fit Erlenmeyer flasks 4 nitrate #1 test tablets, from home test kit, in foil container 4 nitrate #2 test tablets, from home test kit, in foil container Test kit color chart 10-mL graduated cylinder 5 mL 30-ppm aqueous nitrate ion standard solution 5 mL 60-ppm aqueous nitrate ion standard solution 5 mL 90-ppm aqueous nitrate ion standard solution 5 mL 120-ppm aqueous nitrate ion standard solution Centrifuge Glass stirring rod 4 plastic disposable droppers 2 spectrophotometer cells Spectrophotometer

If you spill on yourself any of the aqueous nitrate ion solutions, or any solutions containing nitrate ion and test tablets, rinse the affected area with a large volume of tap water. If you get powder from either test tablet on yourself, rinse the affected area with a large volume of tap water. All liquid wastes can be discarded in a sink and flushed down the drain with a large volume of tap water. During the steps described below when you are shaking the flask after adding one or both nitrate test tablets, you may want to wear disposable gloves to protect your hands.

Obtain two clean spectrophotometer cells. Rinse one cell with several milliliters of distilled water, discard the rinsing, and fill the cell two-thirds full with distilled water. This cell will have only distilled water in it for the duration of the experiment.

Add 5 mL of the 120-ppm aqueous nitrate ion standard solution, measured using the clean, dry 10-mL graduated cylinder, to a clean, dry 50-mL Erlenmeyer flask. Record the actual concentration shown on the label of the bottle. Add one Nitrate #1 test tablet to the solution in the 50-mL Erlenmeyer flask; avoid skin contact with the tablet, if possible. Stopper the flask with a cork stopper, and shake flask and contents until the solid has dissolved. Unstopper the flask, add one Nitrate #2 test tablet (avoid skin contact with the tablet) to the solution, restopper, and shake vigorously for exactly one minute. Let the stoppered flask and contents stand undisturbed for five minutes to insure that the reaction has gone to completion. During the five-minute waiting period, rinse the 10-mL graduated cylinder with several portions of distilled water and discard each rinsing. After the five minutes are over, transfer the contents of the 50-mL Erlenmeyer flask to a clean, dry 13 x 100 mm (or larger) test tube, and centrifuge the test tube and contents to give a clear liquid above any undissolved solids.

6-2 EXPERIMENT 6 SPECTROPHOTOMETRIC DETERMINATION OF NITRATE ION CONCENTRATION

After centrifuging, handle the test tube gently so that none of the solid becomes resuspended. Record the color of the resultant solution. Compare the solution color with the test kit color chart, and record the nitrate ion concentration indicated by the chart.

Use a clean plastic disposable dropper to transfer 2.0 mL of the colored solution in the 13 x 100 mm test tube into the rinsed 10-mL graduated cylinder, and add enough distilled water to give a total liquid volume of 5.0 mL. Mix the contents of the graduated cylinder with a clean glass stirring rod to give the solution referred to hereafter as the dilute colored solution.

Rinse the second spectrophotometer cell with 1 mL of the dilute colored solution you just prepared, discard the rinsing, and fill the cell two-thirds full with the dilute colored solution. Follow your instructor's directions for the operation of your spectrophotometer. Record the absorbance of the solution from 360nm to 600nm at 20nm intervals. Determine the wavelength for each peak observed; you may want to check at 10nm intervals on either side of the wavelength where a peak is first seen to determine the wavelength of the peak more precisely. Discard the contents of the cell, and any dilute colored solution remaining in the graduated cylinder, rinse the cell with two portions of distilled water, and discard each portion. Rinse the 10-mL graduated cylinder with two portions of distilled water, and discard the rinsings.

Repeat the above procedure for preparing the dilute colored solution (including recording the color of the solution before dilution and determining the nitrate ion concentration indicated by the chart) and carrying out the spectroscopy study, substituting the 90-ppm, 60-ppm, and 30-ppm aqueous nitrate ion standard solutions for the 120-ppm solution. Use a different 50-mL Erlenmeyer flask and 13 x 100 mm test tube for each nitrate ion solution. Since you do not know if the color does or does not change on standing for an extended length of time, do not begin preparing a new colored solution until you have completed the spectroscopic measurements with the previously prepared solution. After you have completed the spectroscopic measurements, the distilled water in the one cell can be discarded and the cells returned. To complete the clean-up, discard the mixtures remaining in the 13 x 100 mm test tubes, rinse each test tube and 50-mL Erlenmeyer flask with several milliliters of distilled water, discard the rinsings, and return all glassware.

CALCULATIONS

Plot absorbance (along the y-axis) versus wavelength (along the x-axis) for each of the four colored solutions and draw a smooth curve through the points for a solution. Plot all absorption spectra on the same piece of graph paper. Indicate the nitrate ion concentration in the solution to which each spectrum applies.

Construct a calibration curve by plotting absorbance versus concentration; use the absorbance values for the 30-ppm, 60-ppm, 90-ppm, and 120-ppm solutions at the wavelength you selected. State the wavelength you selected. Draw the best straight line through the points.

For each standard solution, compare the concentration found using the test kit color chart and the actual concentration on the label of the bottle.

Can nitrate ion concentrations up to 140 ppm be determined spectrophotometrically using the reagents from the home test kit? Justify your decision. If so, at what wavelength should the absorbance measurements be made?

6-3 SPECTROPHOTOMETRIC DETERMINATION OF NITRATE ION CONCENTRATION EXPERIMENT 6

SPECTROPHOTOMETRIC DETERMINATION OF NITRATE ION CONCENTRATION

REPORT FORM

Actual Nitrate Ion Color from Chart Concentration Wavelength(s) Where Concentration from Chart Peak(s) Occur

For each standard solution, compare the concentration found using the test kit color chart and the actual concentration.

6-4 EXPERIMENT 6 SPECTROPHOTOMETRIC DETERMINATION OF NITRATE ION CONCENTRATION

Solution Absorbance

Wavelength (nm) 120 ppm 90 ppm 60 ppm 30 ppm

Wavelength selected for constructing the calibration curve

6-5 SPECTROPHOTOMETRIC DETERMINATION OF NITRATE ION CONCENTRATION EXPERIMENT 6

Can nitrate ion concentrations up to 140 ppm be determined spectrophotometrically using the reagents from the home test kit? Justify your decision. If so, at what wavelength should the absorbance measurements be made?

6-6 EXPERIMENT 6 SPECTROPHOTOMETRIC DETERMINATION OF NITRATE ION CONCENTRATION

NOTES TO INSTRUCTOR

The directions in the Experimental Procedure section for preparing the colored solutions were written assuming that the color chart and test reagents from the DRY-TAB Nitrate Test Kit (Product No. 66) from Aquarium Pharmaceuticals, Inc., P.O. Box 218, Chalfont, PA 18914 would be used. Included in the DRY-TAB Nitrate Test Kit are the color chart for determining concentration in ppm, 20 Nitrate #1 test tablets individually sealed in a foil strip, and 20 Nitrate #2 test tablets in a second foil strip; the kit costs approximately $11. Replacement packages, costing approximately $8, contain only 20 Nitrate #1 and 20 Nitrate #2 test tablets packaged in foil strips, so the kit must be purchased to obtain the color chart; do not discard the color chart. The color chart shows a yellow solution color for 0 ppm nitrate ion, an orange color for intermediate concentrations, and a red color for 140 ppm. There is an expiration date printed on the bottom of the box and on each of the foil strips.

If you use a home test kit from another manufacturer, the printed directions supplied in that kit for the preparation of the colored solution must be substituted for the directions for preparing the solutions in this experiment; also, check the ppm range, because you may need to change the concentrations of the standard solutions.

Sodium nitrate (FW=84.99) can be used as the nitrate ion source. Avoid contact of solid sodium nitrate with anything combustible. If you spill the solid or an aqueous sodium nitrate solution on your skin, rinse the affected area with a large volume of tap water. Pour waste aqueous sodium nitrate solutions down the drain and then flush with tap water. An aqueous sodium nitrate solution having a nitrate ion concentration

of 120 ppm is approximately 0.0019 M NaNO3. To prepare 500 mL of the 120-ppm nitrate ion solution: transfer 0.0821 g of solid NaNO3 to a clean 500-mL volumetric flask, add some distilled water to dissolve the solid, fill to the calibration mark with distilled water, and invert several times to mix the contents of the flask. The density of this solution is 1.00 g/mL at 25NC. To calculate the actual nitrate ion concentration in ppm of this solution:

- 62.00g NO3 [mass of NaNO3 x ——————] (1) 84.99g NaNO3 Concentration in ppm = —————————————— x 106 500g solution

The solutions of lower nitrate ion concentration are prepared by diluting the 120-ppm solution with distilled water. The density of each of these solutions is 1.00g/mL, so solution volume prepared and solution mass are equal in magnitude. To prepare 100 mL of the 90-ppm nitrate ion solution, transfer 75.0 mL (measured using a clean, dry 100-mL graduated cylinder) of stock 120-ppm solution to a clean 100- mL volumetric flask, fill to the calibration mark with distilled water, and invert several times to mix. To calculate the actual nitrate ion concentration in ppm in this solution:

actual ppm in stock solution x volume transferred in mL (2) concentration in ppm = ——————————————————————— 100 mL

6-7 SPECTROPHOTOMETRIC DETERMINATION OF NITRATE ION CONCENTRATION EXPERIMENT 6

To prepare 100 mL of 60-ppm and 30-ppm solutions, follow the procedure given above for the 90-ppm solution, but use 50.0 mL and 25.0 mL, respectively, of the stock 120-ppm solution.

Your students may find that it is easier to directly read percent transmittance, %T, from the spectrophotometer rather than absorbance, A. If so, %T can be read and A then calculated using

A = 2.000 - log (%T) (3)

Whether the students work individually or in groups and the number of solution spectra that an individual or group records are determined by the number of available spectrophotometers and the length of time allotted for lab work. The experimental procedure and the materials listed below assume that each student does all parts of the experiment individually; for this option, approximately 2½ hours are needed to complete the lab work. Another option is that each student in a group of four prepares one of the dilute colored solutions and records its spectrum (if possible, each group member uses the same spectrophotometer); spectroscopic data are exchanged among the four students in the group.

With each of the four nitrate ion standard solutions, dissolution of the Nitrate #1 tablet gives a slightly cloudy yellow solution. Especially with the lower nitrate ion concentrations, there may be some solid present after addition of the Nitrate #2 tablet and vigorously shaking for one minute. To prevent the solid from affecting the absorbance (or percent transmittance) readings, each reaction mixture is centrifuged so that only clear liquid is used to prepare the dilute colored solution for the spectroscopic study. A 13 x 100 mm test tube, used in centrifuging the reaction mixture, is very similar in size to the stoppered tube supplied in the home test kit. Some of the original reaction mixtures give very large (>1.5) absorbance readings over the entire wavelength range studied and the absorbance readings do not greatly change. To facilitate reading either A or %T values from the instrument, each reaction mixture is diluted.

The spectrum for the dilute colored solution for each nitrate ion standard solution shows a peak at approximately 430nm. The absorbance at the peak is approximately the same in each of the four solutions, so this peak cannot be used as the wavelength at which to monitor absorbance in determining the nitrate ion concentration. The spectrum for the dilute colored solution prepared using the 120 ppm nitrate ion standard solution shows a second peak, at approximately 520nm. However, the spectra for the solutions with lower nitrate ion concentrations show no well-defined peak at this or nearby wavelengths (using a SPECTRONIC® 20 spectrophotometer), and, at 520nm, absorbance and nitrate ion concentration are not directly proportional (i.e., Beer's Law is not obeyed) for the four solutions. What is most important is that the students draw conclusions appropriate for the data they have collected. What the students will likely find is that there is no wavelength, corresponding to a peak in the absorption spectrum, where there is a direct proportionality between absorbance value and nitrate ion concentration. Thus, it would be difficult to construct a calibration curve, and what looks like a promising way to determine the nitrate ion concentration is, in fact, not feasible.

6-8 EXPERIMENT 6 SPECTROPHOTOMETRIC DETERMINATION OF NITRATE ION CONCENTRATION

Reagents and equipment needed:

5 13 x 100 mm test tubes (or larger size) 4 50-mL Erlenmeyer flasks 4 cork stoppers to fit Erlenmeyer flasks 4 nitrate #1 test tablets, from home test kit, in foil container 4 nitrate #2 test tablets, from home test kit, in foil container Test kit color chart 10-mL graduated cylinder 5 mL 30-ppm aqueous nitrate ion standard solution 5 mL 60-ppm aqueous nitrate ion standard solution 5 mL 90-ppm aqueous nitrate ion standard solution 5 mL 120-ppm aqueous nitrate ion standard solution Centrifuge Glass stirring rod 4 plastic disposable droppers 2 spectrophotometer cells Spectrophotometer Box of disposable wipes Box of disposable gloves

6-9 SPECTROPHOTOMETRIC DETERMINATION OF NITRATE ION CONCENTRATION EXPERIMENT 6

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6-10 EXPERIMENT 7

COMPARING THE PERFORMANCE OF TWO SPECTROPHOTOMETERS

INTRODUCTION

A spectrophotometer has five basic parts: a light source, a device that separates the light into its constituent wavelengths, a compartment to hold the sample, a detector that analyzes the light which has passed through the sample, and a readout device to provide data to the instrument's user. In this experiment, you will first build a spectrophotometer. The light source will be a red LED (light emitting diode). Because the red LED emits only a very narrow range of wavelengths, no device for separating light into its constituent wavelengths is needed or included in your instrument. However, your instrument will then be capable of making measurements at only one wavelength. Your sample will be contained in a spectrophotometer cell inside a 35 mm film canister. The detector will be a CdS photocell; it is a device whose resistance depends on the intensity of light striking it. The readout device will be a multimeter displaying a DC voltage. Your spectrophotometer is powered by a 9V battery, so you are in no danger of electrical shock while using your instrument. You will then use your spectrophotometer to measure the percent transmittances (%T) of aqueous copper (II) sulfate solutions of known concentration. Finally, absorbance measurements with the same aqueous copper (II) sulfate solutions will be made using a commercially available spectrophotometer, so that you can compare the performances of the two instruments.

EXPERIMENTAL PROCEDURE

Always wear safety goggles while performing the experiment.

In general, you should exercise care when building circuits. Never connect or disconnect wires with the power on. Always wear eye protection when building electronic circuits, since you may face hazards from improperly wired devices that fail catastrophically or even from flying pieces of wire when cutting wire or from splattering solder. If you feel any device or battery getting warm, disconnect the power, since something is wrong.

Reagents and Equipment Needed

High intensity red LED 30 mL 0.15 M CuSO4 100-ohm resistor 50-mL beaker 1000-ohm resistor 10-mL graduated cylinder 9-V battery 10-mL volumetric flask with stopper 9-V battery clips 2 15 x 125 mm test tubes (or larger size) CdS photocell 2 spectrophotometer cells Multimeter Spectrophotometer Breadboard Sample cell for LED instrument Plastic 35 mm film canister with top

7-1 COMPARING THE PERFORMANCE OF TWO SPECTROPHOTOMETERS EXPERIMENT 7

Technique Options for Building the Circuit

1. Twist the wires together. This method requires no additional equipment or parts, but it provides the least dependable connections and is not recommended.

2. Use test leads with clips on each end. Much more reliable than twisting the wires together. Additional expense of buying test leads, but they are extremely useful if you plan to do any further circuit construction/testing. As the number of connections you need to make increases the sloppiness of your circuit increases with all of the jumper cables (plus the expense of buying all the cables).

3. Use a breadboard. Easy to use. Initial expense of purchase. Great for quickly trying circuits.

4. Soldering. Best connections. Expense of equipment and supplies. Must learn proper technique. Safety issues. Possible burn hazard. Hazard of breathing fumes during soldering. Always use lead-free solder. Soldering should be done in an area free of combustible/ flammable materials and with proper ventilation. Proper eye protection must be worn and skin should be protected from hot spatters of solder. Instructor must determine if student is capable of safely using this technique.

See "Getting Started in Electronics" by Forrest Mims (approx $3.00 at Radio Shack) for explanation of the above techniques.

Building the LED Spectrophotometer

Wire the circuit as shown in the preceding diagram. You have at least four options (described above) of how to make the electrical connections. The down arrows in the diagram indicate connection to ground. In this case, there is no need for an actual earth-ground connection. All the points with arrows must be connected together. Note that there is a flat spot on one side of the base of the plastic LED housing.

The LED must be wired as shown or it will not produce light. The 100 ohm resister limits the amount of current that flows through the LED. The LED will self-destruct if you connect it to the battery without a current-limiting resistor.

7-2 EXPERIMENT 7 COMPARING THE PERFORMANCE OF TWO SPECTROPHOTOMETERS

Enclose the LED and the photocell in the plastic film canister; see the diagram on page 7-5. You will need to cut holes on opposite sides of the film canister for the red LED and the CdS photocell; make sure that each fits snugly in its hole so that no stray light can enter the film canister. When you build your sample holder using the film canister, you should experiment with your sample container (e.g., the SPECTRONIC® cell or a plastic cuvette) to insure it will fit into the canister between the LED and the photocell. Also, you need to be able to insert and remove the sample container without disturbing the alignment of the LED and the photocell. You may want to use a spectrophotometer cell with a flat bottom for holding the sample.

If you have a setup to solder, you can build the above circuit without the breadboard. If you use the breadboard, you need to become familiar with how the hole patterns interconnect. The line of holes labeled "A" are connected to each other. The column of holes labeled "B" are connected to each other. The column of holes labeled "C" are connected to each other. The column of holes labeled "D" are connected to each other. However, A's, B's, C's, and D's have no connection to each other.

BREADBOARD

A B D

C

Preparation of Solutions of Known Concentration

Obtain 30 mL of approximately 0.15 M CuSO4 in a clean, dry 50-mL beaker. Record the actual solution concentration, which is given on the label of the bottle. Rinse a 10-mL graduated cylinder with several milliliters of 0.15 M CuSO4, and discard the rinsing. Use the graduated cylinder to transfer 2.0 mL of 0.15 M CuSO4 to a clean 10-mL volumetric flask. Record the volume transferred to ±0.1 mL. Add enough distilled water to the 10-mL volumetric flask to fill the flask to the calibration mark. Stopper and invert several times to thoroughly mix the contents of the volumetric flask. Transfer the solution to a clean, dry 15 x 125 mm or larger test tube; label the test tube Solution 1. Rinse the volumetric flask with two portions of distilled water and discard the rinsings. Dispose of all liquid wastes in this experiment

by pouring down the drain and flushing with water. If you spill 0.15 M CuSO4, or any of the solutions prepared from it, on your skin, wash the affected area with a large volume of tap water.

7-3 COMPARING THE PERFORMANCE OF TWO SPECTROPHOTOMETERS EXPERIMENT 7

Prepare Solutions 2 and 3 using 5.0 mL and 7.0 mL, respectively, of 0.15 M CuSO4. Follow the procedure described for Solution 1; the 10-mL graduated cylinder used to measure the 0.15 M CuSO4 does not need to be rinsed between solution preparations. Solution 2 must be transferred to a clean, dry 15 x 125 mm or larger test tube, but Solution 3 can be left in the volumetric flask.

Spectroscopy With Your LED Instrument

Rinse the sample container with distilled water, discard the rinsing, and fill the container with enough distilled water so that the liquid level inside the container is above the top of the LED after the container is inserted into the film canister. (Use this volume of liquid in the sample container for all measurements using your LED instrument.) Place the sample container in the film canister, and measure and record the voltage; this is the measurement for 100%T. Remove the sample container, block the light path (use a piece of black paper or white paper folded many times), and measure and record the voltage; this is the measurement for 0%T. Repeat the pair of voltage measurements with first the sample container containing distilled water, then the path blocked until five of each measurement have been recorded. Discard the water in the sample container.

Rinse the same container with a maximum of 1 mL of Solution 1, discard the rinsing, and fill the container to the proper height with Solution 1. Place the sample container in the film canister, measure and record the voltage, and remove the container from the film canister. The diagram on page 7-5 illustrates the placement of the sample container between the LED and the CdS photocell. Repeat the procedure of inserting the container, recording the voltage, and removing the container until five voltage measurements have been recorded. Transfer the sample of Solution 1 back into the test tube containing

Solution 1. Repeat the procedure described in this paragraph using Solutions 2 and 3 and 0.15 M CuSO4. At the conclusion of this set of measurements, you should have five voltage measurements for each of the

four aqueous CuSO4 solutions.

7-4 EXPERIMENT 7 COMPARING THE PERFORMANCE OF TWO SPECTROPHOTOMETERS

Spectroscopy With a Commercially Available Instrument

Set the instrument at 660nm. Rinse one of the spectrophotometer cells with distilled water, discard the rinsing in a sink, and fill the cell two-thirds full with distilled water. This cell will have only distilled water in it for the duration of the experiment. Rinse the other cell with 1 mL of Solution 1, discard the rinsing, and fill the cell two-thirds full with Solution 1. Follow your instructor's directions for the operation of your spectrophotometer. Record the absorbance of the solution, then remove the cell from the sample compartment. Reinsert the cell in the sample compartment and record the absorbance, until a total of five absorbance readings have been made with Solution 1. Solution 1 can then either be discarded or returned to its test tube. Make five absorbance readings of Solutions 2 and 3, and 0.15 M

CuSO4, using the above procedure. At the conclusion of absorbance value determinations, all solutions can be discarded in a sink and the glassware rinsed with distilled water and returned.

CALCULATIONS

Determine the molar concentration of CuSO4 in Solutions 1-3.

Calculate an average value for the voltage measured with only distilled water in the cell using your instrument; this average voltage is designated V(100). Calculate an average value for the voltage with the light path blocked; this average voltage is designated V(0). For each of the four solutions containing

CuSO4, calculate an average value for the voltage measured with that solution; the average voltage for a solution is designated V(s). The percent transmittance, %T, for a solution is calculated from:

V(0) - V(s) (1) %T = [——————] x 100% V(0) - V(100)

Calculate %T for each CuSO4 solution. The absorbance, A, of the solution can be calculated from:

A = 2.000 - log (%T) (2)

Calculate an average value for the absorbance for each of the four solutions of known molar

concentration of CuSO4.

Compare your LED-based instrument with the commercially available instrument. As part of your comparison, you may want to look at the plot of absorbance versus concentration and draw the best straight line through the data points; this plot is called a calibration curve. The individual data points should lie on or close to the best straight line drawn. Which instrument, yours or the commercial one, would you want to use if you were going to generate data for a calibration curve so you could determine the concentration of the absorbing substance in a solution of unknown concentration of that substance? Briefly explain why you made the choice you indicate.

7-5 COMPARING THE PERFORMANCE OF TWO SPECTROPHOTOMETERS EXPERIMENT 7

COMPARING THE PERFORMANCE OF TWO SPECTROPHOTOMETERS

REPORT FORM

Actual concentration of Stock CuSO4 Solution

Solutions of Known Concentration Data

Solution Volume Transferred (mL) Molar Concentration 1 2 3

Calculations:

7-6 EXPERIMENT 7 COMPARING THE PERFORMANCE OF TWO SPECTROPHOTOMETERS

Spectroscopy Data Using the LED Instrument

Voltage Determination (V) Sample12345Average Water Blocked Solution 1 Solution 2 Solution 3 Stock Solution

Spectroscopy Data Using the Commercial Instrument

Absorbance Measurements Solution12345Average 1 2 3 Stock

7-7 COMPARING THE PERFORMANCE OF TWO SPECTROPHOTOMETERS EXPERIMENT 7

Calculation of A from LED Instrument Data

Solution %T A 1 2 3 Stock

Calculations:

Compare your LED-based instrument with the commercially available instrument. Which instrument would you use to collect data for a calibration curve to be used to determine the solution concentration of an absorbing substance in an unknown? Briefly explain why you made the choice you indicate.

7-8 EXPERIMENT 7 COMPARING THE PERFORMANCE OF TWO SPECTROPHOTOMETERS

NOTES TO INSTRUCTOR

If you don't have a nearby Radio Shack or you want to shop around, then look for an electronics supply house in your yellow pages. Also look for an electronics surplus store. If you know a ham radio operator or can make contact with a local ham radio club, they would be glad to help you find a source of materials, and you probably can also get some help learning to build circuits. There are also numerous mail order firms for ordering electronic parts.

Radio Shack carries a number of introductory electronics textbooks. Your local library is also an excellent source of electronics books.

If the commercial instrument used has a solid state detector or has a wide range photomultiplier tube in it, measuring absorbance at 660nm presents no difficulties. If the instrument has the standard photomultiplier tube, it must be replaced with a red photomultiplier tube and a red glass filter installed (supplied with the red tube) before measurements can be made.

Your students may find that it is easier to directly read percent transmittance from the commercial spectrophotometer rather than absorbance. If so, %T can be read and A then calculated using Equation 2 in the Calculations section.

To prepare 1 L of 0.150 M CuSO4, add 37.4 g of solid CuSO4@5H2O to a 1-L volumetric flask, add some distilled water to dissolve the solid, fill the flask to the calibration mark with distilled water, and mix thoroughly.

Unless you already have a large number of breadboards and multimeters, the cost of materials for building the LED spectrophotometer probably makes this experiment too expensive to be done individually. Students working in groups of 3-4 should be satisfactory. You may want to split the experiment between two lab periods; each group builds a LED spectrophotometer during the first period and then prepares the solutions and carries out the spectroscopic measurements using the two instruments during the second period. Because of the substantial number of voltage measurements to be made using the LED spectrophotometer, you may want to have some members of a group take the voltage readings while the other members obtain the absorbance (or percent transmittance) data for the CuSO4 solutions using the commercial instrument.

The reproducibility of voltage readings, and thus of absorbance values, from the homemade instrument will probably be far worse than measurements made with the commercial instrument.

7-9 COMPARING THE PERFORMANCE OF TWO SPECTROPHOTOMETERS EXPERIMENT 7

Materials needed, per spectrophotometer built:

Part Description Radio Shack Part # Price ($)* High-intensity red LED 276-086 5.19 100-ohm resistor 271-1108 (pack of 5) 0.49 9-V battery 23-553 2.19 9-V battery clips 270-325 (pack of 5) 1.39 CdS photocell 276-1657 (pack of 5) 2.29 1000-ohm resistor 271-1118 (pack of 5) 0.49 Multimeter 22-179 24.99 Breadboard 276-169 21.99

Plastic 35 mm film canister with top

Materials needed, per group:

30 mL 0.15 M CuSO4 50-mL beaker 10-mL graduated cylinder 10-mL volumetric flask with stopper 2 15 x 125 mm test tubes (or larger size) 2 spectrophotometer cells Commercial spectrophotometer Sample container for LED instrument Box of disposable wipes Plastic disposable droppers

*Estimated 1996 prices in U.S. dollars.

7-10 EXPERIMENT 8

FORMULA DETERMINATION BY CONTINUOUS VARIATIONS

INTRODUCTION

Aqueous solutions containing copper (II) ion usually are light blue, due to the presence of the complex 2+ 2+ ion Cu(H2O)6 in the solution. (Two of the six H2O molecules are weakly attached to Cu and these two H2O are sometimes not included in the formula for the complex ion.) Addition of ethylenediamine, H2NCH2CH2NH2 (hereafter designated by en), to an aqueous solution containing copper (II) ion causes the solution color to intensify and change to violet, due to substitution of en for bound H2O molecules. The chemical equation for the reaction is:

2+ 2+ Cu (H2O)6 (aq) + x en(aq) Cu (en)x(H2O)6-2x(aq) + 2x H2O() (1)

The purpose of this experiment is to determine the value for x in Equation 1, using the method of continuous variations.

The method of continuous variations, also known as Job's method, is an experimental procedure for determining the stoichiometry of a pair of reactants in a chemical reaction. The method involves performing a set of trials in which the number of moles of each of the two reactants of interest is systematically varied from one trial to the next while holding constant the total number of moles of reactants used. Experimentally this is done by using the same molar concentration of solute in each stock solution which supplies a reactant, and increasing the volume of one of the stock solutions while decreasing the volume of the other, and keeping the total volume of solution constant. (For example, 1.0 2+ mL of 0.04 M Cu(H2O)6 solution and 9.0 mL of 0.04 M en solution are combined in one trial, 2.0 mL of 2+ Cu(H2O)6 solution and 8.0 mL of en solution in another trial, etc.)

In each trial, a measurable property showing the amount of reaction is monitored. Such properties include mass of precipitate, temperature change, and solution absorbance. The number of moles of the limiting reagent added determines the value of the property. For a reaction with two reactants, the set of 2+ trials performed includes some in which one of the reactants (Cu(H2O)6 in this experiment) is the limiting reagent and others where the second reactant (en here) is limiting. For those trials in which the same reactant is limiting, increasing the number of moles of that reactant increases the value of the monitored property. The maximum value of the monitored property occurs when stoichiometrically equivalent amounts of the two reactants are combined.

To determine the stoichiometry in the chemical reaction of interest, the value of the monitored property is plotted versus the volume of one of the stock solutions. A best straight line is drawn through the points where one of the reactants of interest is the limiting reagent; these are the points where the volumes of the stock solution supplying that reactant were small and the value of the monitored property increases as the volume of the stock solution added increases.

8-1 FORMULA DETERMINATION BY CONTINUOUS VARIATIONS EXPERIMENT 8

A second best straight line is drawn through the points where the other reactant is limiting. This is

illustrated in Figure 1. The ratio of the coefficients e and j in the hypothetical reaction eE(aq) + jJ(aq) qQ(s) is determined by measuring the mass of solid Q which forms when 0.1 M solutions containing E and J are combined. The total solution volume in each trial is 10.0 mL. E is the limiting reagent when 4.0 mL of 0.l M E is added.

Figure 1. Continuous Variations Plot for

eE(aq) + jJ(aq) qQ(s)

The intersection of the two straight lines occurs at the equivalence point where stoichiometrically equivalent amounts were combined. The volume of one stock solution at the intersection point is read from the graph and the volume of the other stock solution can be calculated by difference since the total solution volume is constant in all the trials. In Figure 1, the volumes at the equivalence point are 4.0 mL of 0.l M E and 6.0 mL of 0.1 M J. For each reactant, the volume obtained from the graph and the molarity of solute in the stock solution are used to calculate the actual number of moles of the reactant at the equivalence point. The ratio of the actual number of moles of each of the two reactants at the equivalence point equals the ratio of the stoichiometric coefficients in the chemical equation for the reaction. For the example shown in Figure 1, moles J reacted/moles E reacted = j/e; the volumes at the equivalence point and the 0.l M solute concentrations give a ratio of 3 to 2 for j to e. In this experiment, using Equation 1, this ratio is:

moles en reacted x (2) ———————————— = — 2+ moles Cu(H2O)6 reacted 1

8-2 EXPERIMENT 8 FORMULA DETERMINATION BY CONTINUOUS VARIATIONS

2+ The property monitored in this experiment is the absorbance of Cu(en)x(H2O)6-2x in solution. Assuming 2+ Beer's Law is obeyed by an aqueous solution of Cu(en)x(H2O)6-2x, the relationship between the 2+ absorbance A of the solution and the molar concentration of Cu(en)x(H2O)6-2x in the solution is given by:

2+ A = ab[Cu(en)x(H2O)6-2x] (3)

2+ where a is the molar absorptivity of the aqueous solution containing Cu(en)x(H2O)6-2x and b is the path length; if the same wavelength and sample cell are used for all spectroscopic measurements, ab is a constant and the absorbance of the solution is directly proportional to the molarity of the light-absorbing 2+ substance. After combining the aqueous solutions which supply Cu(H2O)6 and en, the molar 2+ concentration of the Cu(en)x(H2O)6-2x formed in the reaction shown in Equation 1 can be monitored by measuring the percent transmittance, %T, of the solution, from which absorbance can be calculated using

100% (4) A = log ——— %T

EXPERIMENTAL PROCEDURE

Always wear safety goggles while performing this experiment.

Reagents and Equipment Needed

2+ Stock Cu solution (approximately 0.04 M CuCl2) 50-mL beaker 10-mL graduated cylinder 2 10-mL volumetric flasks with stoppers Stock en solution (in hood; approximately 0.04 M) Cuvette 100-mL beaker Watch glass Spectrophotometer Plastic disposable dropper

2+ 2+ 2+ The source of Cu(H2O)6 is the stock Cu solution. Record the concentration of the stock Cu solution. Your laboratory instructor will post a list of volumes of the stock Cu2+ solution that the class will investigate. Select two volumes of stock Cu2+ solution to use for your trials, making sure that you and your classmates choose all possible volumes. (It is acceptable to have more than one student using a particular volume of stock Cu2+ solution, as long as each different volume is used by at least one student.) At the end of the lab period, obtain data from the other students so that you have data over the entire range of volumes.

8-3 FORMULA DETERMINATION BY CONTINUOUS VARIATIONS EXPERIMENT 8

In a clean, dry 5- mL beaker, place about 10 mL more of the stock Cu2+ solution than the sum of the volumes needed for your two trials. Rinse a 10-mL graduated cylinder with a 1-2 mL portion of the stock Cu2+ solution; discard the rinsing down a drain and flush with a large volume of water. If you spill any of the stock Cu2+ solution on your skin, rinse the affected area with a large volume of water. Use the 10-mL graduated cylinder to add one of your selected volumes of stock Cu2+ solution to one clean 10-mL volumetric flask and the other selected volume to the second clean 10-mL volumetric flask. Record each volume added to ±0.1 mL. In a hood, add enough stock en solution (recording its concentration) to each 10-mL volumetric flask to fill the flask to the calibration mark. Stopper the flasks and invert them several times to thoroughly mix their contents. Be careful when using the stock en solution; avoid breathing the fumes, and if you spill any stock en or Cu2+-en solution on your skin, wash the affected area with a large volume of water. Keep the bottle containing the stock en solution capped except when removing solution from it.

Rinse a cuvette with 1-2 mL of the darker, more intensely colored solution you prepared. Pour the rinsing into a 100-mL beaker to collect for later disposal; cover the beaker with a watch glass. This waste must be collected, rather than simply poured down the drain, because of the presence of en. Fill the cuvette about two-thirds full with the more intensely colored solution and clean and dry the outside of the cuvette with a disposable wipe. Using a spectrophotometer, record the percent transmittance, %T, to ±0.1%, from 400nm to 600nm at 20nm intervals. Use a second cuvette two-thirds filled with distilled water for the 100%T adjustment of the instrument at each wavelength. Remember to also set the 0%T adjustment at each wavelength with nothing in the sample compartment and the compartment cover closed. Determine the wavelength where the minimum %T value (i.e. maximum absorbance value, A) is observed; check at 10nm intervals on either side of the wavelength where the smallest %T value is first seen to determine the wavelength of minimum %T more precisely. Record that wavelength. Pour the solution from the cuvette into your waste collection beaker; retain this cuvette for later use with the second solution you prepared, the stock Cu2+ solution, and the stock en solution.

Set the spectrophotometer at the wavelength where the minimum %T was observed. Perform the 0%T and 100%T adjustments of the spectrophotometer at this wavelength. Rinse the cuvette with 1-2 mL of your second solution and pour the rinsing into your waste collection beaker. Fill the cuvette approximately two- thirds full with the solution, wipe the outside of the cuvette with a disposable wipe, and record %T to ±0.1%. After measuring %T, pour the solution from the cuvette into your waste collection beaker. Rinse the cuvette with 1-2 mL of distilled water, discard the rinsing down the drain, and flush with a large volume of water.

Rinse the cuvette with 1-2 mL of the stock Cu2+ solution; discard the rinsing down the drain and flush with a large volume of water. Fill the cuvette about two-thirds full with the stock Cu2+ solution, clean and dry the outside of the cuvette with a disposable wipe, and record %T to ±0.1%. Pour the solution from the cuvette down the drain and flush with a large volume of water. Rinse the cuvette with three 1-2 mL portions of distilled water and discard each rinsing down a drain. Fill the cuvette approximately two-thirds full with the stock en solution, clean and dry the outside of the cuvette with a disposable wipe, and record %T to ±0.1%. Pour the solution from the cuvette into your waste collection beaker. Rinse the cuvette with 1-2 mL of distilled water, and discard the rinsing down the drain and flush with a large volume of water.

Pour any leftover solution from the volumetric flasks into the waste bottle provided. Also pour the wastes collected in your waste collection beaker into the waste bottle. All of the glassware can be rinsed with water and the rinsings flushed down the drain with water.

8-4 EXPERIMENT 8 FORMULA DETERMINATION BY CONTINUOUS VARIATIONS

CALCULATIONS

For each trial (yours and your classmates’), calculate the absorbance A from the measured percent transmittance %T, using Equation 4. Plot A versus the volume of one of the stock solutions for each trial. 2+ Draw one best straight line through the points where Cu(H2O)6 is the limiting reagent and a second best straight line through the points where en limits. Determine the volumes of stock Cu2+ solution and stock 2+ en solution at the intersection point of the straight lines. Calculate the number of moles of Cu(H2O)6 and moles of en at the equivalence point. Determine the value of x using Equation 2.

8-5 FORMULA DETERMINATION BY CONTINUOUS VARIATIONS EXPERIMENT 8

FORMULA DETERMINATION BY CONTINUOUS VARIATIONS

REPORT FORM

Concentration of stock Cu2+ solution

Concentration of stock en solution

Cu2+-en Solutions You Prepared

Solution Solution Volume Used Stock Cu2+ En

Absorption Spectrum of Cu2+-en Complex Ion

Wavelength %T Wavelength %T

Wavelength selected for measuring %T

%T for: less intensely colored Cu2+-en solution

stock Cu2+ solution

stock en solution

8-6 EXPERIMENT 8 FORMULA DETERMINATION BY CONTINUOUS VARIATIONS

In those trials where en is the limiting reagent, do you need to be concerned about the absorbance by 2+ excess Cu(H2O)6 in the solution? Using your experimental results, justify your conclusion.

2+ In those trials where Cu(H2O)6 is the limiting reagent, do you need to be concerned about the absorbance by excess en in the solution? Using your experimental results, justify your conclusion.

Data for Graph

Volume Volume Volume Volume of Cu2+ of en %T A of Cu2+ of en %T A Solution Solution Solution Solution

Volumes at intersection point

Value of x in Equation 1

Calculations:

8-7 FORMULA DETERMINATION BY CONTINUOUS VARIATIONS EXPERIMENT 8

NOTES TO INSTRUCTOR

2+ The value of x is 2 in Cu(en)x(H2O)6-2x. The wavelength where the minimum %T occurs is 540nm.

Copper (II) chloride dihydrate and ethylenediamine are widely available; possible suppliers of these reagents include Aldrich Chemical, Fisher Scientific, Janssen Chimica, Sigma Chemical, and VWR Scientific.

Copper (II) nitrate trihydrate, Cu(NO3)2@3H2O or copper (II) sulfate pentahydrate, CuSO4@5H2O, can be 2+ used as the Cu source if CuCl2@2H2O is not available. To prepare 1 L of 0.0400 M CuCl2, add 6.82 g of CuCl2@2H2O to a 1-L volumetric flask, add distilled water to dissolve the solid, fill with distilled water to the calibration mark on the flask, and mix thoroughly. Each student will need about 20 mL of 0.0400 M

CuCl2.

Ethylenediamine is corrosive; do all preparations of 0.0400 M en in a hood and wear protective gloves. To prepare 1 L of 0.0400 M en, weigh 2.40 g en using a balance in a hood, or measure 2.7 mL en in a 10 mL graduated cylinder, and transfer the liquid to a 1-L volumetric flask. Add enough distilled water to dissolve the ethylenediamine, then fill the flask to the mark with distilled water and mix thoroughly. Pure ethylenediamine reacts with carbon dioxide in air; keep the bottle capped when not using the reagent. The aqueous solution of ethylenediamine is basic and must also be kept in a closed container to avoid exposure of the solution to carbon dioxide. Store 0.0400 M en in a plastic bottle because the solution attacks a glass container.

Any volume of the stock Cu2+ solution between, and including, 1.0 mL and 9.0 mL can be used. A possible set of volumes of the stock Cu2+ solution is: 1.0 mL, 1.5 mL, 2.0 mL, 2.5 mL, etc. A trial using less than 1 mL of the stock Cu2+ solution is not suggested because of difficulty in accurately and precisely measuring this volume. A trial using less than 1 mL of the stock en solution is not suggested because a small error in the volume of this solution can create a large percentage error in the small absorbance value for this solution.

There are several reasons for having each student do only two volume combinations. The volume of liquid waste which must later be treated is minimized while assuring that, even for small lab sections, a sufficient number of data points will be generated. Each trial of a volume combination generates 10 mL of waste solution. If each student did all trials, using integral volumes between 1 mL and 9 mL, 90 mL of waste solution would be generated. The experiment is relatively quick to perform because of the limited number of trials. The experiment design forces students to work together. They must organize themselves to insure that as many volume combinations as possible are used and that duplication is minimized. They must share their experimental results with each other. This might involve writing volume and %T results for each trial on a blackboard so everyone can copy the results from all trials, or having all the students write their results on a sheet of paper and photocopying the page for each student, or entering the results from each trial onto a computer spreadsheet and printing out the data for each student.

The equilibrium constant for the reaction shown in Equation 1 is 1.1x1020. For those trials where 2+ 2+ Cu(H2O)6 is the limiting reagent, there is essentially no Cu(H2O)6 present, and en does not absorb at 2+ the wavelength used in this experiment, so the absorption of light is due entirely to Cu(en)2(H2O)2 . Where en is limiting, essentially all en is complexed with Cu2+ and, potentially, the solution contains two

8-8 EXPERIMENT 8 FORMULA DETERMINATION BY CONTINUOUS VARIATIONS

2+ 2+ colored complexes—Cu(H2O)6 and Cu(en)2(H2O)2 —which can absorb light. The molar absorptivities 2+ 2+ -1 -1 -1 -1 of Cu(en)2(H2O)2 and Cu(H2O)6 at 540nm are about 80 M -cm and 0.2 M -cm , respectively. Even in the trial in which 9 mL of 0.0400 M CuCl2 and 1 mL of 0.0400 M en are combined (giving the 2+ smallest concentration of product and largest concentration of excess Cu(H2O)6 ) to give equilibrium 2+ 2+ concentrations of 0.0020 M for Cu(en)2(H2O)2 and 0.034 M for Cu(H2O)6 , the absorbance due to 2+ Cu(H2O)6 (less than 0.0l for a path length of 1 cm) is not significant compared to the absorbance due to 2+ 2+ Cu(en)2(H2O)2 (about 0.16). Consequently, absorbance by excess Cu(H2O)6 in the trials where en is limiting is not a problem and no correction needs to be applied to subtract out absorbance due to excess 2+ Cu(H2O)6 .

For a total volume of 10 mL of solution prepared in each trial, the intersection of the two straight lines on the graph should occur at 3.3 mL of Cu2+ solution and 6.7 mL of en solution combined. If only integral volumes (1 mL, 2 mL, etc.) of the stock Cu2+ solution are used, fewer volume combinations establish the position of the line where Cu2+ is limiting than volume combinations that establish the position for the second line, where en limits. (There are simply fewer integral values between 1 and 3.3 than between 3.3 and 9 mL.) If only a few non-integral volumes of the stock Cu2+ solution are used, along with all possible integral volumes, it is better to use volumes where Cu2+ limits, to increase the number of points which determine the position of its line.

Using a 10-mL volumetric flask eliminates the need to carefully measure the volume of ethylenediamine solution used. If no 10-mL volumetric flasks are available, prepare each solution in a large test tube and thoroughly mix the solution with a clean glass stirring rod after combining the components of the

solution. If a 10-mL volumetric flask is not used, the volumes of 0.0400 M CuCl2 and 0.0400 M en combined in each solution preparation must each be measured to at least ±0.1 mL and you must assume that the volumes of solutions can be added to give the total volume of the solution prepared. Either a 10- mL graduated pipet or a 50-mL buret can be used instead of the 10-mL graduated cylinder to measure the

volume of 0.0400 M CuCl2 used to prepare the solutions. The 10-mL pipet or the buret must be rinsed with 1-2 mL of 0.0400 M CuCl2 and the rinsing flushed down the drain with a large volume of water.

2+ The solutions containing Cu(en)2(H2O)2 are collected in a waste bottle at the conclusion of the 2+ experiment. To treat the solution containing Cu(en)2(H2O)2 , pour it into a large beaker in a hood. Add to the beaker a volume of vermiculite equal to or larger than the solution volume and cover the beaker with a watch glass. Allow the complex ion to be absorbed into the vermiculite; you may need to wait overnight. Absence of color in the liquid remaining or complete absence of liquid means all of the 2+ Cu(en)2(H2O)2 has been absorbed. In a hood, transfer the vermiculite to a capped container for storage until disposal of the waste.

8-9 FORMULA DETERMINATION BY CONTINUOUS VARIATIONS EXPERIMENT 8

Each student needs the following amounts of reagents and equipment:

20 mL of 0.0400 M CuCl2 50-mL beaker 10-mL graduated cylinder 2 10-mL volumetric flasks with stoppers 20 mL of 0.0400 M en Cuvette 100-mL beaker Watch glass to cover 100-mL beaker 2 disposable plastic droppers for solution transfers

The class as a whole needs:

Bottles of 0.0400 M en in hood One cuvette with distilled water for each spectrophotometer Spectrophotometer(s) One box of disposable wipes per spectrophotometer 2+ 2-L bottle to collect wastes containing Cu(en)2(H2O)2

8-10 EXPERIMENT 9

DETERMINATION OF THE SOLUBILITY PRODUCT OF COPPER (II) IODATE

INTRODUCTION

Aqueous equilibria is an important topic in chemistry. One example is the equilibrium between ions in solution and excess solid in a saturated aqueous solution of a strong electrolyte. The equilibrium condition is described by the solubility product expression. For copper (II) iodate, the chemical equation for the equilibrium of interest is:

2+ - Cu(IO3)2(s) W Cu (aq)+ 2 IO3 (aq) (1) and the solubility product expression is:

2+ - 2 Ksp = [Cu ][IO3 ] (2)

The purpose of this experiment is to determine the numerical value of the solubility product, Ksp, for copper (II) iodate at room temperature. A saturated aqueous solution of the compound is produced by adding excess solid to water. The saturated solution is then separated from the undissolved solid by filtration. The concentration of copper (II) ion in the saturated solution is determined by spectrophotometric analysis using a Beer's Law plot. Data for the Beer's Law plot are obtained from solutions prepared by dilution of an aqueous stock solution of known concentration of copper (II) ion. The copper (II) ion concentration determined from the Beer's Law plot is used to calculate the equilibrium concentration of copper (II) ion in the saturated copper (II) iodate solution. Because the copper (II) and iodate ions are produced from dissolving solid copper (II) iodate in water, the equilibrium concentrations of copper (II) and iodate ions must be in the l:2 ratio shown by the coefficients for the ions in Equation 1. The equilibrium iodate ion concentration is thus twice the equilibrium copper (II) ion concentration. The value for Ksp can then be calculated by substitution of the equilibrium ion concentrations into the solubility product expression, Equation 2.

2+ Aqueous solutions containing copper (II) ion usually are light blue, due to the presence of Cu(H2O)6 in 2+ solution. (Two of the six H2O molecules are weakly attached to Cu and these two H2O are sometimes not included in the formula for the complex ion. To simplify Equations 1 and 2 above, the six H2O molecules bound to copper (II) ion have not been shown.) In the saturated solution of copper (II) iodate, 2+ the concentration of Cu(H2O)6 is too small to produce an easily observed color and to give a reliable percent transmittance or absorbance value when measured with a spectrophotometer such as the SPECTRONIC 20.

9-1 DETERMINATION OF THE SOLUBILITY PRODUCT OF COPPER (II) IODATE EXPERIMENT 9

Addition of ethylenediamine, H2NCH2CH2NH2 (hereafter designated by en) to an aqueous solution containing copper (II) ion causes the color to intensify and to change to violet, due to the formation of 2+ Cu(en)2(H2O)2 via substitution of two en for four of the bound H2O molecules. (The two remaining H2O 2+ 2+ molecules in Cu(en)2(H2O)2 are very weakly attached to Cu and are often not shown in the formula.) The chemical equation for the reaction is:

2+ 2+ Cu(H2O)6 (aq) + 2 en(aq) W Cu(en)2(H2O)2 (aq) + 4 (3) H2O(R)

The equilibrium constant at 25°C for Equation 3 is 1.1 x 1020. For the concentrations of the copper (II) 2+ ion in aqueous solution employed in this experiment, the reaction of Cu(H2O)6 and en thus goes 2+ essentially to completion so that the copper (II) ion in solution is present as Cu(en)2(H2O)2 after addition of en.

EXPERIMENTAL PROCEDURE

Always wear safety goggles while performing the experiment.

Reagents and Equipment Needed

Solid Cu(IO3)2@H2O 6 15 x 125 mm test tubes 50-mL Erlenmeyer flask 6 corks for test tubes

Magnetic stirring bar Solid CuCl2@2H2O Magnetic stirrer 25-mL volumetric flask with stopper 1 sheet 12.5-cm Whatman No. 1 filter paper Analytical balance 75 mm stemless funnel Cuvette Iron ring Spectrophotometer Ring stand 100-mL beaker 50-mL beaker Watch glass 10-mL graduated cylinder Plastic disposable droppers 10-mL volumetric flask with stopper Thermometer 0.10 M en (in hood)

Preparation of Saturated Copper (II) Iodate Solution

Add 0.10 g of copper (II) iodate monohydrate, Cu(IO3)2@H2O, 15 mL of distilled water, and a magnetic stirring bar to a clean 50-mL Erlenmeyer flask. If you spill any of the copper (II) iodate source on your skin, wash the area with a large volume of water. Stir the mixture for 15 minutes at room temperature, using a magnetic stirrer. If no stirrer is available, frequently swirl the contents of the Erlenmeyer flask over the 15 minute period. Record the room temperature.

9-2 EXPERIMENT 9 DETERMINATION OF THE SOLUBILITY PRODUCT OF COPPER (II) IODATE

Flute a piece of 12.5-cm Whatman No. 1 filter paper and place it in a stemless or short-stemmed funnel supported by an iron ring attached to a ring stand. Pour the contents of the Erlenmeyer flask into the fluted filter paper, and collect the filtrate in a clean, dry 50-mL beaker. If the filtrate is not completely free of solid copper (II) iodate, filter the filtrate using the same filter paper and collect using another clean, dry 50-mL beaker.

Preparation of the Copper (II) Iodate Solution for Spectrophotometric Analysis (hereafter referred to as the spectrophotometric solution)

Rinse a 10-mL graduated cylinder with 1-2 mL of your filtrate from the saturated copper (II) iodate solution; pour the rinsing down the drain and flush with water. Use the graduated cylinder to transfer 5.0 mL of the filtrate to a clean 10-mL volumetric flask. Record the volume of solution transferred to ±0.1 mL. In a hood, use the 10-mL graduated cylinder provided with the 0.10 M en (en = ethylenediamine) to add 1 mL of 0.10 M en to the 10 mL volumetric flask. Stopper the flask immediately after adding the 0.10 M en. Be careful when using the 0.10 M en; avoid breathing the fumes and if you spill any 0.10 M en on your skin, wash the affected area with a large volume of water. Keep the bottle containing 0.10 M en capped except when removing solution from it. Add enough distilled water to the 10-mL volumetric flask to fill the flask to the calibration mark. Stopper the volumetric flask and invert it several times to thoroughly mix the contents. Transfer the solution to a clean, dry, labeled 15 x 125 mm or larger test tube; stopper the test tube with a cork stopper. Rinse the 10-mL volumetric flask with two portions of distilled water; pour the rinsings down the drain and flush with water.

2+ Preparation of 0.0100 M Cu (aq) Stock Solution

You will prepare a 0.0l00 M solution of copper (II) ion to use as the stock solution for further dilutions in the preparation of a Beer's Law plot. The source of copper (II) ion for the solution is copper (II) chloride dihydrate, CuCl2@2H2O. Calculate the mass of copper (II) chloride dihydrate needed to prepare 25 mL of 0.0100 M CuCl2. Use an analytical balance which reads to ±0.0001g to weigh the solid. Record the actual mass of the solid used to prepare the solution. Copper (II) chloride dihydrate is a skin and mucous membrane irritant. Avoid breathing the dust, and if you spill any solid or solution on your skin, rinse the affected area with a large volume of water. Use a 25-mL volumetric flask for preparing the solution. Add the solid copper (II) chloride dihydrate to the flask, and add distilled water to bring the solution volume to the calibration mark. Stopper and invert to mix the contents of the volumetric flask thoroughly.

Preparation of Solutions for Generating the Beer's Law Plot

Rinse the 10-mL graduated cylinder (the one previously used to prepare the spectrophotometric solution) 2+ with 1-2 mL of the 0.0100 M Cu (aq) stock solution; pour the rinsing down the drain and flush with water. 2+ Use the graduated cylinder to transfer 1.0 mL of the 0.0100 M Cu (aq) stock solution to the clean 10-mL volumetric flask (also previously used to prepare the spectrophotometric solution). Record the volume of 2+ 0.0100 M Cu (aq) stock solution transferred to ±0.1 mL. In a hood, use the 10-mL graduated cylinder provided with the 0.10 M en to add 1 mL of 0.10 M en to the 10-mL volumetric flask, and stopper the flask immediately after the addition. Again, remember to be careful when using the 0.10 M en. Add enough distilled water to the 10-mL volumetric flask to fill the flask to the calibration mark. Invert to thoroughly mix the contents of the 10-mL volumetric flask. Transfer the solution to a clean, dry 15 x 125 mm or larger test tube; stopper the test tube with a cork stopper and label the test tube Solution 1.

9-3 DETERMINATION OF THE SOLUBILITY PRODUCT OF COPPER (II) IODATE EXPERIMENT 9

Rinse the 10-mL volumetric flask with two portions of distilled water; pour the rinsings down the drain and flush with water.

Prepare Solutions 2,3,4, and 5 using 2.0 mL, 3.0 mL, 4.0 mL, and 5.0 mL, respectively, of 0.0100 M 2+ Cu (aq) stock solution. Follow the procedure described for Solution 1; the 10-mL graduated cylinder used 2+ to measure the 0.0100 M Cu (aq) stock solution does not need to be rinsed between solution preparations.

Spectroscopy

Rinse a cuvette with 1-2 mL of Solution 5. Pour the rinsing into a 100-mL beaker to collect for later disposal; cover the beaker with a watch glass. This waste must be collected, rather than simply poured down the drain, because of the presence of en. Fill the cuvette about two-thirds full with Solution 5, and clean and dry the outside of the cuvette with a disposable wipe. Using a spectrophotometer, record the percent transmittance, %T, to ±0.1%, of Solution 5 from 400nm to 600nm at 20nm intervals. Use a second cuvette two-thirds filled with distilled water for the l00%T adjustment of the instrument at each wavelength. Remember to also set the 0%T adjustment at each wavelength, with nothing in the sample compartment and the compartment cover closed. Determine the wavelength where the minimum %T value (i.e. maximum absorbance value, A) is observed; you may want to check at l0nm intervals on either side of the wavelength where the smallest %T value is first seen to determine the wavelength of minimum %T more precisely. Pour the Solution 5 sample in the cuvette back into the test tube containing Solution 5. Rinse the cuvette with 1-2 mL of distilled water, and discard the rinsing in the sink.

Set the spectrophotometer at the wavelength where the minimum %T was observed with Solution 5, and record that wavelength. Perform the 0%T and 100%T adjustments of the spectro- photometer at this wavelength. Record %T, to ±0.1%, for Solutions 1-4 and for the spectrophotometric solution. For each of these %T measurements, rinse the cuvette with 1-2 mL of the solution to be measured, pour the rinsing into the 100-mL beaker used earlier, fill the cuvette approximately two-thirds full with the solution, and record %T; start with Solution 1. After measuring %T, pour the solution from the cuvette back into its test tube. Before measuring %T for the spectrophotometric solution, first rinse the cuvette with 1-2 mL of distilled water (discard the rinsing in the sink), then rinse with 1-2 mL of the solution.

Clean-up

Wastes from the preparation of saturated copper (II) iodate solution: Give the filter paper with the undissolved copper (II) iodate to your lab instructor for disposal. Flush any remaining filtrate down the drain with a large volume of water.

2+ Waste from the preparation of 0.0100 M Cu (aq) stock solution can be flushed down the drain with a large volume of water.

Wastes from spectroscopy: Pour leftover Solutions 1-5 and the spectrophotometric solution into the waste bottle provided. Also, pour the rinsing wastes collected in the 100-mL beaker into the waste bottle. The glassware can be rinsed with water and the rinsings flushed down the drain with a large volume of water.

9-4 EXPERIMENT 9 DETERMINATION OF THE SOLUBILITY PRODUCT OF COPPER (II) IODATE

CALCULATIONS

Generation of the Beer's Law Plot

Use the mass of copper (II) chloride dihydrate you recorded to calculate the actual molar concentration of 2+ 2+ copper (II) chloride, and thus of Cu (aq), in the 0.0100 M Cu (aq) stock solution you prepared. Use that actual molar concentration to calculate the molar concentration of copper (II) ion (present as 2+ 2+ Cu(en)2(H2O)2 (aq)) in each of the five solutions where 0.10 M en and 0.0100 M Cu (aq) stock solution were combined. Calculate the absorbance for each of the five solutions used to generate the Beer's Law plot and the absorbance of the spectrophotometric solution from their measured %T values. Absorbance is calculated using 100% (4) A = log (———) %T where %T is the measured percent transmittance.

Construct the Beer's Law plot by plotting absorbance of each numbered solution versus the molar concentration of copper (II) ion in that solution. Draw the best straight line through the data points, including the origin as an additional point.

Calculation of the Ksp Value for Copper (II) Iodate

Use the Beer's Law plot and the absorbance of the spectrophotometric solution to determine the molar concentration of copper (II) ion in that solution. Calculate the molar concentration of copper (II) ion in the saturated aqueous solution of copper (II) iodate, remembering to take into account that the saturated solution was diluted with water and 0.10 M en in the preparation of the spectrophotometric solution. Using the coefficients from Equation 1, calculate the molar concentration of iodate ion in the saturated aqueous solution. Calculate the value for Ksp for copper (II) iodate.

Compare your experimental Ksp value with the literature value. Give a complete reference for the literature value.

9-5 DETERMINATION OF THE SOLUBILITY PRODUCT OF COPPER (II) IODATE EXPERIMENT 9

DETERMINATION OF THE SOLUBILITY PRODUCT OF COPPER (II) IODATE

REPORT FORM

Room temperature

Filtrate volume used to prepare spectrophotometric solution

Volume of spectrophotometric solution prepared

Mass of CuCl2@2H2O used

2+ Number of Solution for Volume of .0100M Cu (aq) Total Volume Solution Generating Beer's Law Plot Stock Solution Used Prepared 1 2 3 4 5

9-6 EXPERIMENT 9 DETERMINATION OF THE SOLUBILITY PRODUCT OF COPPER (II) IODATE

2+ Absorption Spectrum of Cu(en)2(H2O)2 (aq)

Wavelength %T Wavelength %T

Wavelength selected for measuring %T

Solution %T Absorbance 1 2 3 4 5 Spectrophotometric Solution

2+ Actual CuCl2 molar concentration in 0.0100 M Cu (aq) stock solution

Calculations:

9-7 DETERMINATION OF THE SOLUBILITY PRODUCT OF COPPER (II) IODATE EXPERIMENT 9

Solution Molar Concentration of Cu2+ 1 2 3 4 5

Calculations:

Concentration of Cu2+ in spectrophotometric solution from Beer's Law plot

Concentration of Cu2+ in saturated aqueous copper (II) iodate solution

Calculations:

9-8 EXPERIMENT 9 DETERMINATION OF THE SOLUBILITY PRODUCT OF COPPER (II) IODATE

- Concentration of IO3 in saturated aqueous solution

Calculations:

Ksp value

Calculations:

Literature value of Ksp of copper (II) iodate

Reference for literature value:

Comparison of experimental and literature values:

9-9 DETERMINATION OF THE SOLUBILITY PRODUCT OF COPPER (II) IODATE EXPERIMENT 9

NOTES TO INSTRUCTOR

Copper (II) chloride dihydrate, ethylenediamine, and sodium iodate (see below) are widely available; possible suppliers of these reagents include Aldrich Chemical, Fisher Scientific, Janssen Chimica, Sigma Chemical, and VWR Scientific.

Copper (II) iodate monohydrate can be obtained from Pfaltz and Bauer, Waterbury, CT. However, considering the small amount of solid needed (0.10 g per person), it may be less expensive and more convenient to prepare the solid yourself. The compound can be easily prepared from copper (II) chloride dihydrate and sodium iodate. For a theoretical yield of 0.0l00 moles (4.31g) of Cu(IO3)2@H2O (FW = 431.3 g/mol): Dissolve 1.71g (0.0100 mol) CuCl2@2H2O in 5 mL of distilled water in a 25-mL Erlenmeyer flask. Dissolve 4.00g (0.0202 mol, a slight excess) NaIO3 in 60 mL of distilled water in a 125-mL Erlenmeyer flask. Add a magnetic stirring bar to the NaIO3 solution and begin stirring. Add the CuCl2 solution slowly to the NaIO3 solution while continuing to stir. A light turquoise precipitate will immediately form. Stir for 15 minutes after adding all of the CuCl2 solution to ensure completeness of reaction. Isolate the solid by vacuum filtration. If the filtrate has a noticeable blue color, transfer the filtrate back into the 125-mL Erlenmeyer flask, add 1.00g NaIO3 to the flask, and stir for 15 minutes. The solid NaIO3 will dissolve and additional product will separate from solution during stirring. Collect this solid by vacuum filtration and combine it with the solid previously isolated. The filtrate can be flushed down the drain with a large volume of water. Allow the solid to air-dry for 2-3 days. The yield of product (a light turquoise powder) is 4.09g (94.9%).

Copper (II) nitrate trihydrate, Cu(NO3)2@3H2O or copper (II) sulfate pentahydrate, CuSO4@5H2O, can be 2+ used as the Cu source if CuCl2@2H2O is not available. To prepare 25 mL of 0.0100 M CuCl2, 0.0426 g of CuCl2@2H2O is needed. If you want to provide the 0.0100M CuCl2 rather than have each student prepare it, add 1.705g CuCl2@2H2O to a 1-L volumetric flask, add distilled water to dissolve the solid, fill with distilled water to the calibration mark on the flask, and mix thoroughly to give 1 L of 0.0100 M

CuCl2. Each student will need approximately 20 mL of 0.0100 M CuCl2.

Ethylenediamine (en) is corrosive; do all preparations of 0.10 M en in a hood and wear protective gloves. To prepare 1 L of 0.10 M en, weigh 6.01 g en using a balance in a hood, or measure 6.7 mL en in a 10- mL graduated cylinder and transfer the liquid to a 1-L volumetric flask. Add enough distilled water to dissolve the ethylenediamine, then fill the flask to the calibration mark with distilled water and mix thoroughly. Pure ethylenediamine reacts with carbon dioxide in air; keep the bottle capped when not using the reagent. The aqueous solution of ethylenediamine is basic and must also be kept in a closed container to avoid exposure of the solution to carbon dioxide. Store 0.10 M en in a plastic bottle to avoid having the basic solution attack its glass container.

Using a 10-mL volumetric flask eliminates the need to carefully measure the volumes of ethylenediamine solution and distilled water used to prepare each solution used in the Spectroscopy section. If there are a sufficient number of 10-mL volumetric flasks available that each student can have six flasks, Solutions 1- 5 and the spectrophotometric solution can be left in the flasks in which they were prepared and do not need to be transferred to stoppered test tubes. If no 10-mL volumetric flasks are available, prepare each solution in a large test tube, and thoroughly mix the solution with a clean glass stirring rod after combining all components for the solution. If a 10-mL volumetric flask is not used, the volumes of the source of copper (II) ion, ethylenediamine solution, and distilled water combined in each solution preparation must each be measured to at least ±0.1 mL, and you must assume that the volumes of

9-10 EXPERIMENT 9 DETERMINATION OF THE SOLUBILITY PRODUCT OF COPPER (II) IODATE

solutions can be added to give the total volume of the solution prepared. A 5-mL pipet (and bulb) can be used, instead of the 10-mL graduated cylinder, to measure the volume of filtrate from the saturated aqueous copper (II) iodate solution used to prepare the spectrophotometric solution. The pipet must be rinsed with 1-2 mL of the filtrate; the rinsing can be flushed down the drain with a large volume of water. Either a 10-mL graduated pipet or a 50-mL buret can be used instead of the 10-mL graduated cylinder to 2+ measure the volume of 0.0100 M Cu (aq) stock solution used to prepare each of the five solutions for the 2+ Beer's Law plot. The 10-mL pipet or the buret must be rinsed with 1-2 mL of 0.0100 M Cu (aq) stock solution and the rinsing flushed down the drain with a large volume of water.

The copper (II) iodate which did not dissolve is separated by gravity filtration from the filtrate. Each student is instructed to give the filter paper with solid collected to the lab instructor. Allow several days for the solid and filter paper to dry in a hood. After the solid is dry, it can be transferred from the filter paper to a capped container for storage until the solid copper (II) iodate (recovered as the monohydrate) is needed when the experiment is performed again. The filter paper can be discarded in the trash.

2+ The solutions containing Cu(en)2(H2O)2 are collected in a waste bottle at the conclusion of the 2+ experiment. Ethylenediamine was not added to the entire 25 mL of 0.0100 M Cu (aq) stock solution in 2+ order to minimize the volume of solution containing Cu(en)2(H2O)2 that requires treatment at the end of 2+ the experiment, and to allow part of the 25 mL of 0.0l00 M Cu (aq) stock solution to be flushed down the 2+ drain with a large volume of water. To treat the solution containing Cu(en)2(H2O)2 , pour it into a large beaker in a hood. Add to the beaker a volume of vermiculite equal to or larger than the solution volume, 2+ and cover the beaker with a watch glass. Allow the Cu(en)2(H2O)2 to be absorbed into the vermiculite; you may need to wait overnight. Absence of color in the liquid remaining or complete absence of liquid 2+ means all of the Cu(en)2(H2O)2 has been absorbed. In a hood, transfer the vermiculite to a capped container for storage until disposal of the waste.

Excess ethylenediamine is added in preparing Solutions 1-4 and the spectrophotometric solution. This 2+ excess, in addition to the large formation constant for Cu(en)2(H2O)2 , insures that essentially all of the 2+ copper (II) ion present is found as Cu(en)2(H2O)2 in each of these solutions. Solution 5 uses a stoichiometric amount of ethylenediamine, if 1 mL of 0.10 M en is added. For this solution only, the volume of 0.10 M is added is crucial and at least 1 mL of 0.10 M en must be added in preparing this 2+ solution. Otherwise, not all copper (II) ion will be present as Cu(en)2(H2O)2 , and the calculated copper 2+ (II) ion concentration will be larger than the actual concentration of Cu(en)2(H2O)2 in solution. Also, solid copper (II) iodate monohydrate is soluble in 0.10 M en. The filtrate from the saturated solution must be completely free of undissolved solid, or the solid will dissolve when the spectrophotometric solution is prepared. If the solid dissolves, this gives a solution higher in copper (II) ion concentration than should be the case, and thus leads to a calculated Ksp value that is too large.

The wavelength where the minimum %T (i.e., maximum A) occurs is 540nm.

The literature value of Ksp for copper (II) iodate at 25°C given in the 74th Edition of the Handbook of -8 Chemistry and Physics, page 8-49, is 6.94 x 10 ; this value is calculated from ªfG° values. The value found in older editions of the Handbook of Chemistry and Physics, for example the 62nd Edition, page B- 242, is 1.4 x 10-7 at 25°C; no indication is given how this value was obtained. The value 7.4 x 10-8 is given in the 14th Edition of Lange's Handbook of Chemistry, p. 8-8. In Volume I of the 4th Edition of Solubilities of Inorganic and Metal Organic Compounds, p. 954, is given an average value of

9-11 DETERMINATION OF THE SOLUBILITY PRODUCT OF COPPER (II) IODATE EXPERIMENT 9

0.00333 mol/L in a saturated aqueous solution of copper (II) iodate; using this concentration gives a Ksp value of 1.5 x 10-7 for copper (II) iodate.

Amounts of reagents and equipment needed, per person:

0.10 g Solid Cu(IO3)2@H2O 6 mL 0.10 M en

0.0426 g Solid CuCl2@2H2O Approximately 100 mL distilled water 50-mL Erlenmeyer flask Magnetic stirring bar Magnetic stirrer 12.5-cm Whatman No. l filter paper 75-mm stemless or short-stemmed funnel Iron ring, to support funnel Ring stand 50-mL beaker 10-mL graduated cylinder 10-mL volumetric flask with stopper 6 15 x 125 mm or larger test tubes 6 cork stoppers to fit test tubes 25-mL volumetric flask with stopper 100-mL beaker Watch glass to cover l00-mL beaker Cuvette 4 disposable plastic droppers for solution transfers Thermometer to record room temperature

Amounts for the class as a whole:

Some extra 50-mL beakers 1 10-mL graduated cylinder and plastic dropper per bottle of 0.10 M en in hood Analytical balance(s) 1 cuvette with distilled water for each spectrophotometer Spectrophotometer(s) 2+ 2-L bottle to collect wastes containing Cu(en)2(H2O)2 1 box of disposable wipes per spectrophotometer

9-12 EXPERIMENT 10

A KINETICS EXPERIMENT: FADING OF PHENOLPHTHALEIN

INTRODUCTION

Phenolphthalein, C20H14O4, is an acid-base indicator frequently used to signal the end point in the titration of a weak acid with a strong base. In an aqueous solution of phenolphthalein having a pH less than or equal to eight, phenolphthalein exists predominantly as the structure shown in Figure 1; this structure is colorless in aqueous solution. As base is added to the solution, raising the pH above eight, reaction between phenolphthalein and hydroxide ion occurs:

- 2- C20H14O4(aq) + 2 OH (aq) C20H12O4 ( aq) + 2 H2O() (1)

The reaction shown in Equation 1 occurs rapidly to produce the structure shown in Figure 2 (only one of the several possible resonance hybrid structures has been drawn); this structure gives a pink-red color to the solution. As more base is added to the aqueous phenolphthalein solution, another reaction occurs:

2- - 3- C20H12O4 (aq) + OH (aq) C20H13O5 (aq) (2)

Figure 3 shows the structure of the product of the reaction shown in Equation 2; this structure is colorless in aqueous solution.

Figure 1. Structure of Figure 2. Structure of Figure 3. Structure of

C20H14O4 in aqueous the anion giving the phenolphthalein in a highly solution at pH 8. pink-red color to an basic aqueous solution. aqueous solution of phenolphthalein.

10-1 A KINETICS EXPERIMENT: FADING OF PHENOLPHTHALEIN EXPERIMENT 10

The pink solution color which occurred when the equilibrium in Equation 1 was established therefore 2- 3- fades to colorless as C20H12O4 is converted to C20H13O5 in the reaction shown in Equation 2. The rate law describing the approach to equilibrium in the reaction shown in Equation 2 is assumed to have the form:

2- p - q Rate = k[C20H12O4 ] [OH ] (3)

The purpose of this experiment is to learn about the kinetics for the reaction shown in Equation 2 and to determine the values for the orders p and q, and the value for the rate constant k, in the rate law shown in Equation 3. Since the reaction shown in Equation 2 is sufficiently slow, and since only one of the substances in the reaction is colored in aqueous solution, the kinetics of this reaction can be studied using 2- visible spectroscopy to monitor the concentration of C20H12O4 over time.

Five trials using different initial hydroxide ion concentrations are performed. In each trial, a small volume of phenolphthalein solution is added to a solution having a substantial hydroxide ion concentration; immediately after combining the two solutions, phenolphthalein is primarily in the form 2- C20H12O4 . The hydroxide ion concentration remains essentially constant over the time period when 2- 3- C20H12O4 is converted to C20H13O5 . As a result, the rate law shown by Equation 3 can be rewritten as:

2- p Rate = kobs[C20H12O4 ] (4)

where the experimentally determined rate constant, kobs, is given by:

- q kobs = k[OH ] (5)

2- If the order with respect to C20H12O4 is one (i.e. if p = 1 in Equation 3 or 4) and if trials are performed using a large hydroxide ion concentration, such that the rate law is given by Equation 4, the reaction is said to show overall pseudo first order kinetics. The integrated rate law obtained from Equation 4 is:

2- 2- ln [C20H12O4 ] = -kobst + ln [C20H12O4 ]0 (6)

2- 2- 2- where [C20H12O4 ] is the molar concentration of C20H12O4 at time t after mixing and [C20H12O4 ]0 is the 2- molar concentration of C20H12O4 at time t = 0.

2- Assuming Beer's law is obeyed by an aqueous solution of C20H12O4 , the relation between the absorbance 2- A of the solution and the molar concentration of C20H12O4 in the solution is given by:

2- A = ab[C20H12O4 ] (7)

2- where b is the path length and a is the molar absorptivity of the solution containing C20H12O4 for a specific wavelength; if the same wavelength and sample cell are used for all spectroscopic measurements, ab is constant.

10-2 EXPERIMENT 10 A KINETICS EXPERIMENT: FADING OF PHENOLPHTHALEIN

Absorbance can be calculated using:

100% (8) A = log (———) %T where %T is the experimentally measured percent transmittance.

2- If At represents the absorbance due to C20H12O4 at time t and A0 is the absorbance at time t = 0, substitution of Equation 7 into Equation 6 and simplification gives:

ln At = -kobst + ln A0 (9)

The plot of ln At versus time will give a straight line with the slope of the line equal to -kobs. One can, 2- therefore, determine if the reaction is first order with respect to C20H12O4 by plotting ln At versus time for each trial performed and seeing if each plot gives a straight line.

To determine the order with respect to hydroxide ion, Equation 5 is manipulated to give

- ln kobs = ln k + q ln [OH ] (10)

- A plot of ln kobs from each trial versus ln [OH ] for that trial is a straight line, with the slope of the line equal to q, the order with respect to hydroxide ion, and the y-intercept equal to ln k, from which k can be calculated.

Each reactant in Equation 2 is negatively charged and in aqueous solution is surrounded by a combination of solvent molecules and other ions collectively referred to as an ionic atmosphere. The number of ions in the ionic atmosphere increases with increasing ionic strength. Increasing the ionic atmosphere tends to reduce electrostatic repulsions between two like-charged reactants, so collision of the two reactant ions is enhanced, which appears in the rate law as an increase in the value of k. This dependence of k on ionic strength is a complication in the present experiment, since it has been assumed that differences in rate between trials are due only to differences in hydroxide ion concentration. Thus, the ionic strength must be kept constant from trial to trial. In this experiment, constant ionic strength is achieved by using 0.25 M NaCl to dilute the stock 0.25 M NaOH in preparing the less concentrated NaOH solutions. Aqueous NaCl solutions are colorless, so there is still only one colored substance, 2- C20H12O4 , in solution in each of the trials. Under the reaction conditions used, chloride ion does not react with any other substance in solution.

The wavelength of light to be used for the measurements is determined from the visible spectrum of a solution prepared by adding an aqueous phenolphthalein solution to a pH l0 buffer solution. The fading of phenolphthalein occurs so slowly at this pH that there is no change in the spectrum during the time required to record the spectrum.

10-3 A KINETICS EXPERIMENT: FADING OF PHENOLPHTHALEIN EXPERIMENT 10

EXPERIMENTAL PROCEDURE

Always wear safety goggles while performing the experiment.

Reagents and Equipment Needed

10 mL pH 10 buffer solution 3 100-mL beakers 50-mL beaker 2 watch glasses to cover 100-mL beakers 0.5% Phenolphthalein Plastic disposable droppers Cuvette 10-mL volumetric flask with stopper Spectrophotometer 10-mL graduated cylinder 35 mL 0.25 M NaCl Thermometer 40 mL 0.25 M NaOH 1.0 M HCl

Obtain 10 mL of the pH 10 buffer solution in a clean 50-mL beaker. Add one drop of the 0.5% phenolphthalein solution to the beaker to give the buffer-phenolphthalein solution; swirl to mix. (If you get any of this solution on your skin, wash the affected area with a large volume of water.) Rinse a cuvette with 1-2 mL of this solution and discard the rinsing in the sink. Fill the cuvette about two-thirds full with buffer-phenolphthalein solution and clean and dry the outside of the cuvette with a disposable wipe. Using a spectrophotometer, record the percent transmittance, %T, to ±0.1%, for the buffer- phenolphthalein solution from 400 nm to 600 nm at 20-nm intervals. Use a second cuvette filled with distilled water for the 100%T adjustment at each wavelength. Remember to also check the 0%T adjustment at each wavelength with nothing in the sample compartment and the compartment cover closed. Determine the wavelength where the minimum %T value (i.e. maximum absorbance value) is observed; check at 10-nm intervals on either side of the wavelength where the smallest %T is seen to get a more accurate value for this wavelength. Use the same spectrophotometer for the remainder of the experiment. Pour the solution in the cuvette back into the beaker; rinse the cuvette with several portions of distilled water and discard the rinsing in a sink. Retain this cuvette for all other %T measurements in this experiment.

Obtain 35 mL of 0.25 M NaCl and 40 mL of 0.25 M NaOH in separate, labeled 100-mL beakers. Cover each beaker with a watch glass. Obtain one clean plastic disposable dropper for dispensing each solution. Rinse a 10-mL volumetric flask with 1-2 mL of 0.25 M NaCl and discard the rinsing in a sink. Rinse a 10-mL graduated cylinder with 1-2 mL of 0.25 M NaOH; pour this rinsing into a 100-mL beaker used to collect wastes for later treatment before disposal. Be careful with the 0.25 M NaOH; if you get any of this solution on your skin, wash the affected area with a large volume of water. Using the 10-mL graduated cylinder, transfer 2.0 mL of 0.25 M NaOH to the 10-mL volumetric flask; record the volume of 0.25 M NaOH transferred to ±0.1 mL. Fill the 10-mL volumetric flask to the calibration mark with 0.25 M NaCl. Stopper the volumetric flask and invert several times to mix the contents of the flask.

When your spectrophotometer becomes available, check the 0%T and 100%T adjustments, using distilled water in the second cuvette (used earlier) for the 100%T adjustment. Add one drop of 0.5% phenolphthalein to the 10-mL volumetric flask; stopper the flask and invert it several times to mix the

10-4 EXPERIMENT 10 A KINETICS EXPERIMENT: FADING OF PHENOLPHTHALEIN

contents to give Solution 1. Rinse the cuvette with 1-2 mL of Solution 1 and discard the rinsing in the 100-mL waste collection beaker. Fill the cuvette about two-thirds full with Solution 1 and clean and dry the outside of the cuvette with a disposable wipe. Record the %T value, to ±0.1%, for Solution 1; this is the reading at time = 0 seconds. Record the %T value and the elapsed time at 30 second intervals after the initial reading until 6 minutes have elapsed or until the %T value becomes l00%T, whichever occurs first. Remove the cuvette from the spectrophotometer between readings. (The temperature of the solution will increase if the cuvette continuously remains in the light beam of the spectrophotometer. If the solution temperature increases, the rate constant for the reaction studied will increase. In order to obtain valid orders, the value for the rate constant for each trial must remain constant over the course of that trial, and all rate constants must be determined at the same temperature.) At the end of data acquisition for Solution 1, pour the solution from the cuvette and the unused solution left in the volumetric flask into the waste collection beaker. Rinse the cuvette with several portions of distilled water and discard each rinsing in the sink. Rinse the 10-mL volumetric flask with two 1-2 mL portions of 0.25 M NaCl and discard each rinsing in the sink.

Repeat the procedure outlined above for Solution 1 using instead 4.0 mL of 0.25 M NaOH to prepare Solution 2. Repeat again, using 6.0 mL to prepare Solution 3. Repeat once more, using 8.0 mL to prepare Solution 4. At the end of data acquisition for Solution 4, rinse the 10-mL volumetric flask with 1-2 mL of distilled water and discard the rinsing in the sink; then rinse the volumetric flask with 1-2 mL of 0.25 M NaOH and pour the rinsing into the waste collection beaker. Rinse the 10-mL graduated cylinder with several portions of distilled water and discard each rinsing in a sink.

Fill the 10-mL volumetric flask to the calibration mark with 0.25 M NaOH. When your spectrophotometer becomes available, add one drop of 0.5% phenolphthalein to the volumetric flask, stopper it, and mix to produce Solution 5. Acquire the %T and time data. Pour Solution 5 from the cuvette into the waste collection beaker, rinse the cuvette with several portions of distilled water, and discard each rinsing in the sink. Pour the remaining solution in the 10-mL volumetric flask into the waste collection beaker, rinse the flask with several portions of distilled water, and discard each rinsing in the sink.

Record room temperature.

Pour the buffer-phenolphthalein solution from the 50-mL beaker and any remaining 0.25 M NaOH into the l00-mL waste collection beaker. Calculate the volume of 1.0 M HCl which must be added to neutralize 40 mL of 0.25 M NaOH (the products of the neutralization reaction are sodium chloride and water). Add that volume of l.0 M HCl to the waste collection beaker and mix the solutions. Be careful with the l.0 M HCl; if you get any l.0 M HCl on your skin, rinse the area with a large volume of water. After neutralization, the contents of the waste beaker can be discarded down the drain with a large volume of water. Also, any remaining 0.25 M NaCl can be poured down the drain. The 50-mL and 100- mL beakers can be rinsed with water and the rinsings discarded in the sink.

10-5 A KINETICS EXPERIMENT: FADING OF PHENOLPHTHALEIN EXPERIMENT 10

CALCULATIONS

For the numbered solutions, use Equation 8 to calculate the absorbance A from each measured %T value. Calculate ln A for each A, plot ln A versus time, and draw the best straight line through the points. You may wish to put all five plots on the same graph. For each solution, do the points lie on or close to the 2- straight line you drew? What can you conclude about the order for C20H12O4 in the rate law? Briefly justify your conclusion. For each of the five plots, determine the slope of the best straight line and the value for kobs. For each solution prepared by dilution of the stock 0.25 M NaOH with 0.25 M NaCl, calculate the hydroxide ion concentration in the solution. (The volume of one drop of 0.5% phenolphthalein solution is assumed to be insignificant compared to 10 mL.) For each solution, calculate - - ln [OH ] and ln kobs. Plot ln kobs versus ln [OH ] for the five solutions and draw the best straight line through the points; the slope of this line is the order with respect to hydroxide ion and the y-intercept equals ln k. Calculate k from the y-intercept.

10-6 EXPERIMENT 10 A KINETICS EXPERIMENT: FADING OF PHENOLPHTHALEIN

A KINETICS EXPERIMENT: FADING OF PHENOLPHTHALEIN

REPORT FORM

Room temperature

Absorption Spectrum for Buffer-Phenolphthalein Solution

Wavelength %T Wavelength %T

Wavelength selected for measuring %T

Solution Volume of 0.25 M Volume of Solution [OH-] NaOH Used Prepared

Calculations:

10-7 A KINETICS EXPERIMENT: FADING OF PHENOLPHTHALEIN EXPERIMENT 10

Solution 1 Solution 2

%T Time A 1nA %T Time A 1nA

10-8 EXPERIMENT 10 A KINETICS EXPERIMENT: FADING OF PHENOLPHTHALEIN

Solution 3 Solution 4

%T Time A 1nA %T Time A 1nA

10-9 A KINETICS EXPERIMENT: FADING OF PHENOLPHTHALEIN EXPERIMENT 10

Solution 5

%T Time A 1nA

For each solution, do the points lie on or close to the best straight line through the points? What can 2- you conclude about the order for C20H12O4 ? Briefly justify your conclusion.

10-10 EXPERIMENT 10 A KINETICS EXPERIMENT: FADING OF PHENOLPHTHALEIN

- Solution Slope of Best Value of kobs ln [OH ] ln kobs Straight Line 1 2 3 4 5

Calculations:

- Slope of line in ln kobs vs ln [OH ] plot

Calculations:

Order for OH-

- Y-intercept of ln kobs vs ln [OH ] plot

Value of k

10-11 A KINETICS EXPERIMENT: FADING OF PHENOLPHTHALEIN EXPERIMENT 10

NOTES TO INSTRUCTOR

Each student needs less than 0.5 mL total of the 0.5% phenolphthalein. It is convenient for the students if you have a bottle, with dropper, of the phenolphthalein solution by each spectrophotometer. To prepare 100 mL of 0.5 % phenolphthalein, add 0.50 g of solid phenolphthalein to a 100-mL volumetric flask. Add 50 mL of absolute ethanol or 95 % ethanol (either will work) to the flask and swirl to completely dissolve the solid. Then add distilled water to the flask to bring the solution level to the calibration mark on the flask's neck and invert several times to thoroughly mix the flask's contents.

To prepare 1 L of pH 10 buffer solution, combine 3.46 g of solid NaHCO3 and 70 mL (measured with a 100-mL graduated cylinder) of 0.25 M NaOH in a 1-L volumetric flask. If the solid does not completely dissolve in the aqueous NaOH solution, add distilled water and swirl to dissolve the solid. After the solid has dissolved, fill the flask to the calibration mark and mix thoroughly.

To prepare 1 L of 0.25 M NaOH, put 10.0 g of solid NaOH in a 1-L volumetric flask. Add distilled water to dissolve the solid, fill the flask with distilled water to the calibration mark, and mix thoroughly. Be careful with both the solid NaOH and the aqueous solution. If you spill either of them on your skin, rinse the affected area with a large volume of water. You may want to wear gloves while preparing the solution. Solid NaOH absorbs water and reacts with carbon dioxide from the atmosphere; keep the bottle capped when not using the reagent. The aqueous NaOH solution must also be kept in a closed container to avoid exposure of the solution to carbon dioxide. Store 0.25 M NaOH in a plastic bottle to avoid having the basic solution attack its glass container. Solid NaOH is not a primary standard; if a more accurate and precise value for the NaOH concentration than 0.25 M is desired (it is not necessary in this experiment), the aqueous NaOH solution can be standardized by titration using potassium hydrogen phthalate with phenolphthalein as the indicator.

To prepare 1 L of 0.250 M NaCl, add 14.61 g of solid NaCl to a 1-L volumetric flask. Add distilled water to dissolve the solid, fill the flask to the mark with distilled water, and mix thoroughly.

Carry out the preparation of 1.0 M HCl in a hood. If you use 12 M HCl (concentrated hydrochloric acid) as the HCl source, you may want to wear gloves during the preparation. Gaseous HCl has a pungent, irritating odor; avoid breathing the fumes from the 12 M HCl bottle. If you spill any 12 M HCl on your skin, immediately rinse the affected area with water. To make 1 L of 1.0 M HCl, transfer 83 mL of 12 M HCl, measured in a hood with a 100-mL graduated cylinder, to a 1-L volumetric flask one-third filled with distilled water. Fill the flask with distilled water to the calibration mark and mix thoroughly. The function of the 1.0 M HCl is to neutralize NaOH in the solution in the waste collection beaker; 40 mL of 0.25 M NaOH requires 10 mL of 1.0 M HCl.

10-12 EXPERIMENT 10 A KINETICS EXPERIMENT: FADING OF PHENOLPHTHALEIN

Using a 10-mL volumetric flask eliminates the need to carefully measure the volume of 0.25 M NaCl added to 0.25 M NaOH in preparing Solutions 1-4. If no 10-mL volumetric flasks are available, prepare Solutions 1-4 in separate large test tubes and thoroughly mix each solution with a clean glass stirring rod after combining the NaOH and NaCl solutions. The volumes of 0.25 M NaOH and 0.25 M NaCl combined in each solution must be measured to at least 0.1 mL, and you must assume that the volumes of solutions can be added to give the total volume of the solution prepared. A 10-mL graduated pipet with bulb can be used instead of the 10-mL graduated cylinder to measure the volume of 0.25 M NaOH used to prepare Solutions 1-4. The 10-mL pipet must be rinsed with 1-2 mL of 0.25 M NaOH and the rinsing poured into the waste collection beaker for later neutralization before disposal.

The wavelength where the minimum %T (i.e. maximum A) occurs is 550 nm. An initial %T reading between 10%T and 20%T at time t = 0 is desirable in each trial; this may require adding more than one drop of 0.5% phenolphthalein. A small initial %T reading insures that the %T reading will not reach l00%T and that equilibrium in the reaction shown in Equation 2 will not be established before the end of the 6 minute timing interval. A higher initial %T reading is acceptable if a change in %T value is observed in the trial. An initial reading above 70%T should be avoided. For Solution 1, the solution with the smallest hydroxide ion concentration, the %T value does not appreciably change over the timing interval. The %T value increases substantially over the timing period for Solution 5 (largest OH- concentration). Monitoring of %T with time should start immediately after combining the phenolphthalein and NaOH solutions to insure that a sufficient number of data points is collected for the plot; if there is a long delay, it is possible that in the solutions with larger hydroxide ion concentrations, the %T of the solution will become l00% before enough data points have been collected.

The reaction is first order with respect to phenolphthalein and first order with respect to hydroxide ion. An ionic strength of 0.25 is used in each trial.

2- Only a very small concentration of C20H12O4 is needed to give a noticeable color to an aqueous solution. 2- A 0.5% aqueous phenolphthalein solution, the source of C20H12O4 , is approximately 0.015 M in C20H14O4. Only one drop of the 0.5% solution is added to 10 mL of aqueous sodium hydroxide solution in each trial to give the colored solution whose rate of loss of color is studied. The phenolphthalein concentration in the colored solution is much less than the hydroxide ion concentration. The statement in the introduction that the hydroxide ion concentration remains essentially constant is valid.

The equilibria involving phenolphthalein and the anions produced from it in basic solution, and the structures of the anions, are described by Lalanne (J. Chem. Educ. l97l, 48, 266-268). Masood et al. (Chim. Anal. l970, 52, l289-l295) studied the effect of ionic strength on the rate of fading of phenolphthalein. At an ionic strength of 0.14 (the largest they employed in their study), the value found for k was 104.40 x 10-4 L-mol-1-s-1 (this is 0.626 M-1-min-1 ) at 25°C. Nicholson (J. Chem. Educ. l989, 66, 725-726) describes an experiment to study the fading of phenolphthalein; an ionic strength of 0.30 was used and the value found for k at this ionic strength was 1.1 M-1-min-1 at 23°C.

10-13 A KINETICS EXPERIMENT: FADING OF PHENOLPHTHALEIN EXPERIMENT 10

Amounts of reagents and equipment needed, per person, are:

10 mL of pH 10 buffer solution < 0.5 mL of 0.5% phenolphthalein 50-mL beaker Cuvette 3 l00-mL beakers 2 watch glasses to cover 100-mL beakers 40 mL of 0.25 M NaCl 30 mL of 0.25 M NaOH 2 plastic disposable droppers 10-mL volumetric flask with stopper 10-mL graduated cylinder 20 mL of distilled water 10 mL of 1.0 M HCl Thermometer to record room temperature

The class as a whole needs:

Spectrophotometers 1 cuvette with distilled water for each spectrophotometer 1 box of disposable wipes per spectrophotometer 1 bottle with dropper of 0.5% phenolphthalein per spectrophotometer

10-14