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2 The information in this publication is provided for reference only. All information contained in this publication is believed to be correct and complete. Thermo Electron Corporation shall not be liable for errors contained herein nor for incidental or consequential damages in connection with the furnishing, performance or use of this material. All product specifications, as well as the information contained in this publication, are subject to change without notice. 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 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: Technical Support Thermo Electron Corporation (North America, Asia Pacific, Middle East, Africa and Latin America) 5225 Verona Road Madison WI U.S.A. Telephone: or Fax: Careplan.Techsupport@thermo.com Thermo Electron Corporation (Europe) Mercers Row, Cambridge CB5 8HY, UK Telephone: Int +44 (0) Fax: Int +44 (0) Careplan.Techsupport@thermo.com SPECTRONIC, GENESYS and Educator are trademarks of Thermo Electron Scientific Instruments Corporation, a subsidiary of Thermo Electron Corporation Rev. R

3 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.

4 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

5 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.

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7 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 Absorption Spectrum Determination of Concentration Using Beer's Law Importance of Wavelength Selected in the Beer's Law Experiment Red Cabbage ph Indicator Spectrophotometric Determination of Nitrate Ion Concentration Comparing the Performance of Two Spectrophotometers Formula Determination by Continuous Variations Determination of the Solubility Product of Copper (II) Iodate A Kinetics Experiment: Fading of Phenolphthalein

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9 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

10 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

11 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

12 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

13 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

14 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

15 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

16 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

17 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

18 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

19 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

20 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

21 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

22 GENERAL OPERATING INSTRUCTIONS GENESYS 20 SPECTROPHOTOMETER This page intentionally left blank. vi-4

23 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

24 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 setup; the proper alignment is easily lost. 1-2

25 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

26 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 Selection No Yes 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

27 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

28 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

29 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

30 COMPACT DISC (CD) SPECTROPHOTOMETER EXPERIMENT 1 This page intentionally left blank. 1-8

31 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 twothirds 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

32 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

33 EXPERIMENT 2 ABSORPTION SPECTRUM ABSORPTION SPECTRUM REPORT FORM Absorption Spectrum of Red Solution Wavelength (nm) Absorbance Wavelength (nm) Absorbance Wavelength(s) where the peak(s) occur(s) 2-3

34 ABSORPTION SPECTRUM EXPERIMENT 2 Absorption Spectrum of Yellow Solution Wavelength (nm) Absorbance Wavelength (nm) Absorbance Wavelength(s) where the peak(s) occur(s) 2-4

35 EXPERIMENT 2 ABSORPTION SPECTRUM Absorption Spectrum of Diluted Stock Solution Wavelength (nm) Absorbance Wavelength (nm) Absorbance Color of stock solution used to prepare diluted solution Wavelength(s) where the peak(s) occur(s) 2-5

36 ABSORPTION SPECTRUM EXPERIMENT 2 Absorption Spectrum of Orange Solution Wavelength (nm) Absorbance Wavelength (nm) Absorbance Volume of red solution used Volume of yellow solution used Wavelength(s) where the peak(s) occur(s) 2-6

37 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

38 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

39 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 A r = absorbance of stock red solution, A y = absorbance of stock yellow solution, A 0 = absorbance of orange solution, V r = volume of stock red solution used to prepare the orange solution, and V y = volume of stock yellow solution used to prepare the orange solution. At any wavelength, A 0 has contributions from the red and yellow components present. If Beer's Law is obeyed, the volume ratio V r /V y is given by: V r A o - A y (1) = V y A r - A 0 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

40 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 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 = 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