Beer’s Law

The absorbance A is the amount of light that a solution absorbs. For a fixed wavelength of light and
a fixed path length through which the light travels, the absorbance is proportional to the concentration c,

A=Kc

where the constant K may be determined by experiment. The procedure will be to make a solution of
known concentration c and determine its absorbance A. To make sure that Beer's Law applies to the
compound of interest, measurements will be made on five different solutions. Rather than averaging the
results, a least-squares line will be drawn through the data points and the slope of the line will determine
the best value for K. Once K is known, the absorbance of other solutions of the same compound can be
predicted (but note that other compounds will have a different value of K). Alternatively, and more usually,
the unknown concentration of a solution of the compound may be determined by measuring its absorbance
and calculating its concentration using

Although absorbance is directly proportional to concentration, the amount of light that is transmitted
through the solution is usually recorded using a spectrometer. For a given wavelength, the amount of light
that is transmitted through pure water is set to 100%. For a solution of a given compound, the relative
amount of light that is transmitted is recorded as the percent transmittance %T, and the absorbance is
calculated using the formula

We will be using tartrazine, a yellow food dye. Like with most dyes, a little goes a long way. You will
prepare four dilutions from a stock solution of known concentration, measure the percent transmittance of
each solution, calculate the absorbance of each solution, and make a graph to determine the best value of
K. You will then use this graph to determine the percent purity of a solid sample of the yellow sparkles that
are put on cakes.

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By E. Nuckels
 Solution Preparation

1. Remember that we are using a food dye, so a little goes a long way! Approximate the number of
grams needed of tartrazine in order to make 500.0 mL of a 4.00 x 10-5M solution.

2. Determine the exact molarity of actual tartrazine stock solution.

3. Take a 250 mL beaker back to the stock solution bottle and obtain approximately 100 mL of
solution. Return to your station.

4. Use a transfer pipette to transfer 5.0 mL from the 250 mL beaker to a 50.0 mL flask. Add water to
the line on the flask from the wash bottle to create 50.0 mL of solution. Snap the cap on to the
flask. HOLDING THE TOP AND BOTTOM OF THE FLASK, turn the volumetric flask upside down,
allowing the air bubble to travel through the flask to mix the solution. Repeat until all the solution is
homogeneous. Calculate the molarity in your 50.0 mL flask.

5. Use a transfer pipette to transfer 10.0 mL from the 250 mL beaker to a 50.0 mL flask. Add water
from the wash bottle until the volume is 50.0 mL. Mix your solution well. Calculate the molarity in
your 50.0 mL flask.

6. Use a transfer pipette to transfer 25.0 mL from the 250 mL beaker to a 50.0 mL flask. Add water
from the wash bottle until the volume is 50.0 mL. Mix your solution well. Calculate the molarity in
your 50.0 mL flask.

7. Transfer 35.0 mL from the 250 mL beaker to a 50.0 mL flask. Add water from the wash bottle until
the volume is 50.0 mL. Mix your solution well. Calculate the molarity in your 50.0 mL flask.

8. Record your molarities on the table on the last page.

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 Spectrometer Setup

1. A Vernier Spectrometer should be attached to your Vernier Lab Quest. If not, attach it. Turn on
power by depressing silver button at top left of Lab Quest.

2. Use the stylus (attached with a string, may need to pull out from back of Lab Quest) to click on
Mode, then change Full Spectrum to Events with Entry. Click OK.

3. Click on the red box, select Change Wavelength, then set wavelength to 425 nm. Click OK.

4. Click on the red box, select Change Units, then select % Transmittance.

5. Click on red box, select Calibrate, then wait for countdown. Meanwhile, without touching the clear
surfaces, fill your cuvette with distilled water (3/4 full is plenty, but no bubbles), wipe the clear
surfaces with a Kimwipe, and place into the spectrometer (the clear edges should be front and back
to allow light in and out). Click on Finish Calibration once Finish Calibration lights up, then press
OK after OK lights up (may take a little while).

6. From now on, only record the percent transmittance for each solution, don't change any settings.

7. Empty the cuvette into a temporary waste container. Use a plastic pipette to add a little of your
most dilute solution to the cuvette, then place in the waste container, twisting so as to clean all
sides. Repeat the cleaning process two more times, then fill the cuvette (3/4 is plenty) with the
most dilute solution. Wipe off the clear sides with the Kimwipe, place in the spectrometer, wait
until the reading stabilizes, then record the percent transmittance on the last page.

8. Cleaning each time, record the percent transmittance of your other four solutions (including the
stock solution).

9. Calculate the absorbance of each solution in the table.

Sparkles

1. Measure out a 0.4g sample of sparkles and note the exact mass on the last page. Add solid to your
250.0 mL volumetric flask. Use a water bottle to wash the last bits off the weigh boat and into the
flask. You can add distilled water quickly until you approach the neck of the flask, but then go more
slowly using a water bottle. Add water to the line (bottom of meniscus at top of line). HOLDING
THE TOP AND BOTTOM OF THE FLASK, turn the volumetric flask upside down, allowing the air
bubble to travel through the flask. Repeat until all the solid has dissolved. Using your wash bottle,
add more water until the flask contains 250.0 mL of solution (volumes don't add, some of the
molecules fit in the holes). Mix your solution well.

2. Clean your cuvette with your Sparkles solution, then record your percent transmittance and
calculate your absorbance.

3. Clean your cuvette with distilled water and dry.

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Name: Partner:

Graphing Data

1. Graph absorbance vs. concentration in Excel for all data points excluding the Sparkles. Type
molarity in the first column, absorbance (not transmittance) in the second column.

2. Highlight the numbers, click on Insert, choose Scatter, click on picture that has Points with no lines,

3. Under Chart Tools, click on the Layout menu and choose More Trendline Options. Use the Linear
regression type, then select Set Intercept and set it equal to zero, select Display Equation on
Chart, then click on Close. Record the value of the constant K.

Results

Sample Molarity %T A = -log(%T/100%)

5.00mL  50.0 mL

10.0 mL  50.0 mL

25.0 mL  50.0 mL

35.0 mL  50.0 mL

Stock solution

Sparkles

Exact mass of Sparkles: g Value of K from graph M-1

Calculations

1. Use the best fit line to determine the molarity of tartrazine in your Sparkles solution.

2. Since you made 250.0 mL of solution, how many moles of tartrazine are in the sample? Grams?

3. Based on the number of grams you measured out, what percent by mass of the sample is
tartrazine?
Molarity of tartrazine in
Sparkles
Mols of tartrazine

g of tartrazine

% tartrazine

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By E. Nuckels