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Experiment #2
Determining the Concentration of an Unknown
Beer’s Law
The primary objective of this experiment is to determine the concentration of an
unknown copper (II) sulfate solution. You will use a Colorimeter (a side view is shown in
Figure 1) to measure the concentration of each solution. In this experiment, red light
from the LED light source will pass through the solution and strike a photocell. A higher
concentration of the colored solution absorbs more light (and transmits less) than a
solution of lower concentration. The Colorimeter monitors the light received by the
photocell as percent transmittance.
Figure 1
You will prepare five copper (II) sulfate solutions of known concentration (standard
solutions). Each solution is transferred to a small, rectangular cuvette that is placed into
the Colorimeter. The amount of light that penetrates the solution and strikes the
photocell is used to compute the absorbance of each solution. When you graph
absorbance vs. concentration for the standard solutions, a direct relationship should
result. The direct relationship between absorbance and concentration for a solution is
known as Beer’s law.
You will determine the concentration of an unknown CuSO4 solution by measuring its
absorbance with the Colorimeter. By locating the absorbance of the unknown on the
vertical axis of the graph, the corresponding concentration can be found on the
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horizontal axis. The concentration of the unknown can also be found using the slope of
the Beer’s law curve.
OBJECTIVES
In this experiment, you will

Prepare and test the absorbance of five standard copper (II) sulfate pentahydrate
solutions.
 Calculate a standard curve from the test results of the standard solutions.
 Test the absorbance of a copper (II) sulfate solution of unknown molar
concentration.
 Calculate the molar concentration of the unknown CuSO 4.5H2O solution.
Prepartion of Solutions (direct method):
In order to calculate the amount (grams) of a compound to use in a solution you need to
know the concentration (mole/L), the volume (V), and the molar mass (g/mole) of the
compound. Remember that Molarity x Volume = moles:
moles x liters x grams
= grams
liter
mol
Let’s say you want to make a 200 ml of a 0.5 M solution of CuSO4.5H2O. The molar
mass(MM) is 249.68 g/mol. How many grams of copper sulfate pentahydrate do you
need to measure out?
M x V x MM = grams
0.5 moles
grams
x 0.200 L x 269.98
mol = 24.968 grams
L
You would then measure out 24.968 g of copper sulfate pentahydrate and bring the
volume of water up to 200 mL for a 0.5000 M solution. Please note that you do not
simply add 200 mL of water. Why? Displacement. 24.968 g is a handful and will
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displace a certain volume of water. Therefore, it will not take exactly 200 mL to make
the solution.
Preparation of Solutions (dilutions):
Suppose that you are given 0.5031 M solution of CuSO4.5H2O and asked to make 50
mL that is 0.2 M! You can’t use the above to figure out how much copper sulfate to
weigh out. So you use something called dilutions. This is a process used to make a
concentrated solution of something less concentrated. For example, you buy Miracle
Grow at Wal-Mart as a concentrated liquid. You put that on your plants and you kill
them. However, if you read the instructions you will see that you should add 10 drops of
Miracle Grow to 1 quart of water- that is a dilution!!!!
When doing dilutions remember, moles before must equal moles after. You are not
changing the number of moles, only the volume that they are in. So, the following must
be true:
Molesbefore = Molaritybefore x Vbefore and Molesafter = Molarityafter x Vafter
This gives us the equation M1V1 = M2V2
Back to the above problem:
Mbefore (or M1) = 0.5031 M
Vbefore( or V1) = ? (what you are trying to solve)
Mafter (or M2) = 0.2 M
Vafter (or V2) = 50 mL
(0.5031 M) x V1 = 0.2M x 50 mL
V1 = 19.88 mL
You would take 19.88 mL of the original solution and dilute with water in a 50 mL
volumetric flask. That gives you a new solution with a molarity of 0.2 M.
Absorption of light by Solutions:
If light is passed through a solution, a certain amount will be absorbed and a certain
amount will be transmitted. Transmittance is simply a ratio of the light shone through the
solution (Io) and the light exiting the sample (I). Values range from 0 to 1 with 1 meaning
100 % transmittance. The spectrophotometer will give you a % T value.
A more common way of measuring light in solutions is absorbance which is defined as
-log T. The absorbance of a solution is related to the concentration of a solution using
Beer’s Law:
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A=εcl
Where
A = absorbance
c = concentration (M)
ε = molar absorptivity (M-1/cm)
l = path length (cm) of sample container (usually taken as 1.0 cm)
Determining concentration from a standard curve:
You have collected the following data in the lab. Based on that data you need to
construct a standard curve and determine the concentration of that unknown from the
standard curve.
Molarity
0.5031
0.3522
0.2516
0.1006
unknown
%T
19.3
32.0
45.3
72.5
33.1
T
0.193
0.320
0.453
0.725
0.331
A
0.714
0.495
0.344
0.140
0.480
Take this data and make a graph in Excel of absorbance (Y-axis) vs molarity (x-axis).
Absorbance
Chart Title
0.8
Absorbance
0.6
0.4
Linear
(Absorbance)
0.2
0
0
0.2
0.4
0.6
concentration
To determine the concentration of the unknown, draw two lines; one from the y-axis to
the line and from the line down to the x-axis. That will be the concentration of your
solution.
Unknown concentration = 0.32 M (from graph above)
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PROCEDURE
1. Obtain and wear safety goggles.
2. Into a plastic weigh boat, weigh approximately accurately 5 grams of powdered
copper(II) sulfate pentahydrate, CuSO4.5H2O. .
Place a small funnel into the neck of a clean 50 mL volumetric flask. Transfer the
CuSO4.5H2O quantitatively to the volumetric flask by carefully raking the solid from
the weigh boat into the funnel and thus into the flask. Do very small amounts at a
time to that the funnel will not get clogged up. After all is transferred use a wash
bottle to add a few milliliter of water to the weight boat and dissolve any residual
solid. Pour this water into the funnel and then use the wash bottle to rinse any solid
on the inside of the funnel into the volumetric flask. Keep the flask on the desk and
do not shake it during this process.
Carefully fill the volumetric flask to the mark, put a stopper in it and shake until all of
the solid is dissolved. Note that the level of liquid is below the mark on the neck.
The sum of the volumes of water and CuSO4.5H2O separately is larger than when
they are mixed. The volumes are not additive.
Carefully fill the flask to the mark again and shake to mix. Calculate the molarity of
the solution (should be about 0.4M). Mark this solution 4.
3. Obtain a 2.0 mL and a 5.0 mL transfer (they have a bulge in the middle and no
graduations) from the soaking apparatus. Rinse them (see procedure below) with
water and then rinse with the above 0.4 M solution using a rubber bulb for suction.
4. Obtain three clean 10 mL volumetric flasks. Into one of them use the 2 mL pipette to
place 2 mL of the above 0.4 M solution. Mark this solution 1. Use the 5 mL pipette
to place 5 mL of the 0.4 M solution into a second 10 mL volumetric flask. Mark this
solution 2. Use both the 2 mL and the 5 mL pipettes to place 7 mL of the 0.4 M
solution into the third 10 mL flask. Mark this solution 3.
Calculate the molarity of these three solutions. They should be about 0.08M, 0.2M
and 0.28 M respectively, the exact values depending on the exact molarity of the
solution being diluted.
The transmittance of the three diluted solutions and the 0.4 M solution will be
measured with the colorimiter.
RINSE THE PIPETS AS FOLLOWS: HOLD THE PIPETTE UPSIDE DOWN AND
PUT THE TIP ON THE SPIGOT OF THE FAUCET. TURN THE WATER ON
CAREFULLY AND ALLOW WATER TO RUN THROUGH THE PIPETTE SLOWLY
TO RINSE IT. THEN RUN A SMALL AMOUNT OF DISTILLED WATER THROUGH
THE PIPETTE TO RINSE OUT THE TAP WATER. RETURN THE PIPETTE (TIP
UP) TO THE DISTILLED WATER SOAK CONTAINER
5. Connect a Colorimeter to Channel 1 of the Vernier computer interface. Connect the
interface to the computer using the proper cable.
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6. Start the Logger Pro program on your computer. Open the file “Experiment #2 Beer’s
Law” from Logger Pro. (This program is now on the desktop under the File “CLAB
124 Experiments”)
7. Calibrate the Colorimeter.
a. Prepare a blank by filling an empty cuvette ¾ full with distilled water. Place the
blank in the cuvette slot of the Colorimeter and close the lid.
b. Set the wavelength on the Colorimeter to 635 nm, press the CAL button. When
the LED stops flashing the calibration is complete.
8. You are now ready to collect absorbance-concentration data for the five standard
solutions.
a. Click
.
b. Remove the cuvette from your Colorimeter and pour out the water. Using
solution 1, rinse the cuvette twice with ~1 mL amounts, and then fill it ¾ full.
Wipe the outside with a tissue, place it in the Colorimeter, and close the lid.
c. When the absorbance readings stabilize, click
, type the concentration of
Test Tube 1 in the edit box, and press the ENTER key. The data pair should now
be plotted on the graph.
d. Discard the cuvette contents as directed. Using solution 2, rinse and fill the
cuvette ¾ full. Wipe the outside, place it in the Colorimeter, and close the lid.
When the absorbance readings stabilize, click
, type the concentration of
Test Tube 2 in the edit box, and press the ENTER key.
e. Repeat the procedure for solutions 3 and 4. Do not test the unknown solution
until Step 9.
f. When you have finished testing the standard solutions, click
.
g. Examine the graph of absorbance vs. concentration. Click the Linear
Regression button, . A best-fit linear regression line will be shown for your five
data points.
9.
Record the absorbance values, for each of the four trials, in your data table.
10. Determine the absorbance value of the unknown CuSO4 solution.
a. Obtain about 5 mL of the unknown CuSO4 in a clean, dry, test tube. Record the
number of the unknown in your Data Table.
b. Rinse the cuvette twice with the unknown solution and fill it about ¾ full. Wipe
the outside of the cuvette, place it into the Colorimeter, and close the lid.
c. Read the absorbance value displayed in the Meter window. (Important: The
reading in the Meter window is live, so it is not necessary to click
to read
the absorbance value.) When the displayed absorbance value stabilizes, record
its value as Trial 6 in your data table.
d. Dispose of any of the remaining solutions as directed.
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DATA TABLE
Trial
Concentration (mol/L)
Absorbance
1
2
3
4
Unknown number ____
DATA ANALYSIS
1. Calculate the linear regression (best-fit line) equation of absorbance vs.
concentration for the five standard CuSO4 solutions. Print a graph showing the data
and linear-regression equation for the standard solutions.
2. Determine the concentration of the unknown CuSO4 solution using the slopeintercept equation.