Indigo

Blue

EXPERIMENT: BEER’S LAW

In this experiment we will be investigating the color produced when light is absorbed by atransparent solution. The color a solution will appear to us can be predicted by using the colorwheel. If the chemicals in the solution absorb only red light, the solution will appear blue-green. Blue-green is the color directly opposite red on the color wheel. Colors opposite one another onthe color wheel are called complementary colors. Red and blue-green are complementary colors. If a solution appears blue then we would find that the chemicals are absorbing orange light sinceblue and orange are complementary. It should be noted that there is no such thing as purple light(purple is red + violet ) so if a solution is green it can’t absorb “purple” light. Instead it absorbsreds and violets.

We would also expect that as the concentration of the absorbing chemicals increase the amountof light absorbed would probably increase also. This relationship is called “Beer’s Law.” Theinstrument we will use to determine the type of light absorbed by the solution and the amount oflight absorbed is called a spectrometer.

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Verification of the Color Wheel

Connect the spectrometer to the USB port of your computer and open Logger Pro. If theinstrument has not been used before on your computer it may take a few moments for thecomputer to install the drivers necessary. Eventually you should see this screen:

Select Experiment from the toolbar and then Calibrate, Spectrometer from themenu. Fill a sample holder (called a cuvette) about 3/4 full of distilled water and position it intothe spectrometer so that the rubbed sides of the cuvette do not interfere with the light beam. Toavoid having fingerprints interfere with the light readings, always handle the cuvettes near the

top. The special Kimwipe tissures can be used to wipe the outside of the cuvettes. Click FinishCalibration and wait until the spectrum calibrates throughout the wavelength range.

You will observe the absorption spectrum of 5 solutions: blue food coloring, yellow foodcoloring, a mixture of blue and yellow food coloring, phenolphthalein in a basic solution, andCu . Fill another cuvette about 3/4 full of blue food coloring. Place the cuvette containing2+

the solution into the spectrometer and press the green Collect button in toolbar. The button

turns red. Your spectrum might look something like the one below. Press Stop to terminatethe run.

Logger Pro is plotting absorbance along the Y axis and wavelength along the X axis. Absorbance is a variable that measures the amount of light the solution absorbs. The greater theabsorbance, the more light of that particular wavelength the solution is absorbing. You mayneed to change the scale on the graph so that the graph fills the greatest area of the screen

possible. To do this either right click on the graph or select Options from the toolbar and then

go to Graph Options, Axes Options. The scaling along the X and Y axes can now bechanged. For this graph, change the Y axis to Autoscale From 0 and leave the default scaling onthe X axis.

This should scale the Y axis so that the peak absorbance if near the top of the graph.

If you want to determine the wavelength of the peak in the spectrum, select Analyze from the

toolbar and then Interpolate.

Use the color wheel along with the color of the light most strongly absorbed to predict thecolor of the solution. In the mixture of blue and yellow there will be two colors that are absorbedabout equally so record both. You can use the colored background in the graph to visuallydetermine the color begin absorbed, however you need to appreciate that it is hard to render exactcolors on a computer. You might also want to use the table below to determine the color of thelight absorbed from the interpolated wavelength. Even in the table, determination of an “exact”color of the light absorbed is not possible since there are an infinite variation in hues aswavelength changes. Compare the predicted color to the color actually observed to verify theprediction is correct (or considering the uncertainties in relating a color to a specific wavelength,at least close). Notice in the chart that there is no wavelength for “purple” light, since purplelight doesn’t exist.

approximate wavelength (nm) color of light

410 violet

430 indigo

480 blue

500 blue-green

530 green

560 lemon-yellow

580 yellow

610 orange

680 red

To measure the spectrum of the next solution store your current trial by selecting

Experiment from the toolbar and then Store Latest Run.

Replace the cuvette with another one containing yellow food coloring, press the green

Collect button, then Stop, and Store the Latest Run. You will notice that once you haverecorded the second solution, both graphs appear. Now pour the blue food coloring and theyellow food coloring into an empty beaker and mix the two. Place a sample of this mixture backinto the spectrometer and record the spectrum. Use the color wheel to explain the resultingspectrum. Remember that there is no such thing as purple light.

At some point your graph may have so many lines on it that it is hard for you to keep track ofwhich line belongs to which substance. You can temporarily remove some of the lines by going

to Graph Options, Axes Options. In the Y-axis column window you can select and deselectthe data you want plotted. .

Using your wash bottle, rinse out the two curvettes with distilled water. Record theabsorption spectra of basic phenolphthalein and Cu and verify that the color wheel predicts2+

their colors. Save your Logger Pro file to your personal laptop or a USB storage device.

The Effect of Concentration on the Spectrum

You will measure the absorbance of three solutions of crystal violet: 1.00 x 10 M crystal-5

violet (CV) and two diluted solutions of crystal violet. To prepare the two diluted solutions, usepipets to add the quantities of solution shown in the table below to initially dry 50 mL beakers. Stir the solutions thoroughly. Assume that the volumes are additive and calculate the molarity ofcrystal violet in each diluted solution.

volume of crystal violet (mL) volume of water (mL)

10.00 5.00

5.00 10.00

For this part of the experiment you are going to change the cosmetics of the graph. Right

clicking on the graph or selecting Options from the tool bar will all you to access the GraphOptions. Remove the Mouse Position and Delta (the numbers that show up in the lower

left hand corner of the graph) and change the Grid so that no line is used on Major and MinorTicks Styles. Next remove background colors on the graph. To remove the background colors

unclick the box labeled Draw Visible Spectrum(Wavelength Graphs).

Place a cuvette containing the CV solution of highest concentration into the spectrometer andrecord the spectrum. Remember that you must store the current run before you can record asecond spectrum. Record the spectra of the other two CV solutions and display all three curveson the axes. When you are finished you should have three graphs displayed on the same set of

axes. Give the graph a title. This is done by going to Graph Options as described previously. The title is usually written (name of Y variable) vs (name of X variable) with some additionalwords added to give clarity to what materials are being used. Finally, interpolate the wavelengththat gives the maximum absorption. You should see that the peaks in each graph occur at thesame wavelength but have lower absorbance values as the concentration decreases. Save yourLogger Pro file again.

Report:

1. Copy the completed graph showing the effects of concentration into your Excelspreadsheet.

Beer’s Law

Record the spectrometer number on your spreadsheet so that you may use the sameinstrument over several weeks. Close the previous Logger Pro file and open an new one. Placethe solution of highest concentration into the spectrometer. Click on the Configure theSpectrometer button in the toolbar. This is the button to the left of the green collect button thatlooks like a graph with a spectrum beneath it. You should see an absorption spectrum for crystalviolet. Change the Collection Mode from Abs vs Wavelength to Abs vs Concentration. Thecomputer will automatically find the wavelength of highest absorption.

Select two additional wavelength, one about 50 nm longer than wavelength at the maximumabsorption and one about 50 nm shorter. The display should look like this except yourwavelengths will be different.

Remove the grid lines and the Mouse Position and Delta as previously described. Youmay wish to change the colors of the variables to more closely match the actual color of lightbeing measured (see the wavelength/color table) or change them all to black. To change the

colors, double click on the column heading for the variable in the table. Select Options fromthe menu and you can change the color used for the variable (as well as the style of pointprotectors and the way the number is written). When selecting colors avoid using very lightcolors since they don’t print very well.

Recalibrate the spectrometer with distilled water. With the cuvette still in the spectrometer start

the data collection by pressing the green Collect button. To the right of that button a Keepbutton becomes active. Press the Keep button (NOT STOP) and enter the concentration ofcrystal violet in the solution (0 since it is distilled water). You can enter the concentrations inscientific notation as 1.00e-5. Do not put any spaces in or use a non-numeric symbol other thanan “e” or “E”. Place a cuvette of crystal violet into the spectrometer. Since the cuvettes fitloosely in the sample holder try to position the cuvettes into the sample holder exactly the same

way each time. Press the Keep button and enter the appropriate concentration. Repeat until all

the solutions of crystal violet have been measured. Then press the red Stop button.

To draw the lines on the graph, go to Analyze in the toolbar and select Linear Fit.

To avoid clutter on the graph the linear fit boxes can either be minimized or they can be hidden

by right clicking on the box and removing the check mark in the Show on Graph option. Eachbox must be hidden individually.

Report:

1. Prepare a graph showing all three sets of data linear fitted but without the linear fit boxesshowing. Give the graph a title and copy to your Excel spreadsheet.

Analysis of the Unknown

Obtain a crystal violet solution of unknown concentration. Place a cuvette of this

solution into the spectrometer and select Analyze from the toolbar followed by

Interpolation Calculator. Choose the wavelength of the graph you want to interpolate. You will need to do this for each of the lines individually.

Report:

2. Copy the graph to your Excel spreadsheet making sure the interpolation boxes areshowing. Try to position the boxes so that they are in blank areas of the graph. Enter theinterpolated concentration into your Excel file

3. To estimate the uncertainty in your interpolated concentration, add a small amount (say

0.005) to each of the absorbance readings that were shown in the interpolationcalculator dialog boxes. Type these new values for absorbance into the interpolationcalculator boxes. Do this for each of the three wavelengths. Copy this graph to yourExcel file.

4. For each wavelength, subtract the two interpolated concentrations. Determine whichwavelength gives the smallest change in concentration. This wavelength should give themost accurate concentration reading. Record the concentration of the unknown as thevalue obtained from the data using this wavelength.

When you are finished, disconnect the spectrometer from your computer. Discard thesolutions you used down the drain. Rinse the cuvettes with water. Be sure to save your LoggerPro file to your computer or USB storage device