Colorimetry
Build and use a colorimeter. *
CONTENTS
1
Objectives ............................................................................................................................................. 1
1.1
Prerequisite Skills and Knowledge ............................................................................................... 1
1.2
Research Skills .............................................................................................................................. 2
1.3
Learning Objectives ...................................................................................................................... 2
2
Pre-Experiment ..................................................................................................................................... 2
2.1
The Colorimeter ............................................................................................................................ 2
2.1.1
Light Transmittance and Absorbance ................................................................................... 3
2.1.2
The Light Source................................................................................................................... 3
2.1.3
The Light Sensor ................................................................................................................... 4
2.2
3
Prepare for the experiment ............................................................................................................ 4
Laboratory Manual................................................................................................................................ 5
3.1
Materials Check Off List............................................................................................................... 5
3.2
Safety and Waste Disposal Protocols............................................................................................ 5
3.3
Experimental Procedure ................................................................................................................ 5
3.3.1
Assemble the Colorimeter ..................................................................................................... 5
3.3.2
Optimize the Light Sensor .................................................................................................... 6
3.3.3
Turn on the Light Source ...................................................................................................... 7
3.3.4
Serial Dilution in Cuvettes .................................................................................................... 7
3.3.5
Measure the Transmittance as a Function of Concentration at 626 nm ................................ 7
3.3.6
Analysis of Transmittance Measurements ............................................................................ 8
3.4
Post-Lab Assignment .................................................................................................................... 8
1
1.1
OBJECTIVES
PREREQUISITE SKILLS AND KNOWLEDGE
Students should have experience building simple circuits from diagrams, writing simple virtual
instruments, and pipetting.
*
The Lego colorimeter was developed by Jonas Asheim, et al., as described in J. Chem. Educ. 2014, 91, 1037-1039.
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1.2
RESEARCH SKILLS
After this lab, students will have had practice in:
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1.3
Using LabVIEW
Programming
Building circuits containing LEDs
Measuring transmission and absorbance of light through a medium
Colorimetry
Serial dilution
LEARNING OBJECTIVES
After this lab, students will be able to:
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Use LabVIEW to display the readings from an instrument connected through the Vernier Sensor
DAQ
Measure the transmittance of a solution
Measure the absorbance of a solution
2
2.1
PRE-EXPERIMENT
THE COLORIMETER
Colorimetry is measurement of the transmittance or absorbance of visible light by a substance in solution.
The term colorimetry comes from the fact that all visible light has color. Thus the substances evaluated by
colorimetry are colored.
In this experiment you will measure the transmittance of colored visible light by a food dye dissolved in
water. You will build your own colorimeter from LEDs, batteries, and basic circuit elements. Lego blocks
will be used to hold everything together.
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Colorimetry |3
2.1.1 Light Transmittance and Absorbance
Transmittance T is the fraction of light at a given wavelength that passes through a light-absorbing
medium. Transmittance is defined as
𝑇 = 𝐼⁄𝐼
0
where I0 is the intensity of the incident radiation entering the medium and I is the intensity of the
transmitted radiation leaving the medium. T is often expressed as percent transmittance, %T = 100(I/I0)
Absorbance is the proportion of light absorbed by a medium. Absorbance is defined as
𝐼
𝐴 = log10 ( 0⁄𝐼 ) = −log10 𝑇
In this experiment, you will find the relationship between transmittance, absorbance, and the
concentration of the solution under study.
2.1.2 The Light Source
The light source is an LED that produces light at 626 nm. That’s red light. Look for the specifications
sheet RedLED in the Colorimetry folder on e-Learning. Recall that LEDs must be connected so that the
current flows through them in a particular direction. What direction? A few hints:
The LED is connected in series with two 1.5 V batteries and a 100 Ω resistor. What will be the current for
this circuit?
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2.1.3 The Light Sensor
LEDs can also be used as light sensors. If you shine a light of the right wavelength at an LED it will
generate a voltage. You will find lots of information if you Google “LED as light sensor”.
2.2
PREPARE FOR THE EXPERIMENT
Read through the entire procedure to prepare you laboratory notebook. You will need to prepare an Excel
spreadsheet to collect and analyze your data. Test your preparation and your spreadsheet with the
problems on the Pre-Experiment Quiz.
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Colorimetry |5
3
3.1
LABORATORY MANUAL
MATERIALS CHECK OFF LIST
Each small group of (2-3) students will have:
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3.2
Laptop computer with LabVIEW.
Set of micropipettes with associated tips
Vernier SensorDAQ.
Differential Voltage Sensor
Current Sensor
1 breadboard with set of 4 – 6 terminal blocks
1 breadboard terminal block removal tool
1 set of jumper cables and wires
2 pigtail alligator clip connectors
2 banana plug connectors w/detachable alligator clips
100 Ω resistor
2 red LEDs (peak = 626 nm)
2 1.5V batteries with battery holders
Lego bricks
o 2 grey bricks 2x1 with hole
o 2 red bricks 2x2
o 2 grey bricks 2x2 with bottom cut off
o 1 grey plate 6x16
6 Cuvettes with internal diameter 10 x 10 mm
Stock solution of blue food color (FD&C Blue #1) at concentration 1.00 AU
DI water dispenser
SAFETY AND WASTE DISPOSAL PROTOCOLS
Cuvettes and pipette tips should be disposed of in the benchtop lab waste container or in the large lab
waste container in the room. Food color solutions may be poured down the drain when no longer needed.
Clean up spills quickly, to avoid electrocution and damage to electronics.
While in the X-Laboratory, do not eat, drink or apply ointments to the skin, even if you are not working
directly with toxic substances.
3.3
EXPERIMENTAL PROCEDURE
3.3.1 Assemble the Colorimeter
Instructions for assembly are given below, followed by comments. Read both instructions and comments
before assembling the various parts.
3.3.1.1 Light Source Assembly
Press a red LED fully into the hole of a “brick 2x1 with hole”. The corner of another Lego brick can be
used as a tool. Place the “brick 2x1 with hole” over a red “brick 2x2”.
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3.3.1.1.1 Comments
1. It is important that the LED cannot move in the hole when connected to the circuit, otherwise the light
that reaches the detector will vary. This is easily achieved if the LED is forced into the brick with a
decent push.
2. Once the LED is inserted it is hard to remove without breaking.
3. Insert the LED into the hole with the legs horizontal and the long leg always to the right. Use a
sharpie to mark the anode side of the LED by putting a + on that side of the brick with hole.
4. Gently bend the legs away from each other. (See Diagrams 1 and 2)
Build the light source circuit following Circuit Diagram 1, below. Use two 1.5 V batteries for the 3V and
a 100 Ω resistor. Add a switch to the circuit and leave the switch open, for now.
Circuit Diagram 1
3.3.1.2 Light Sensor Assembly
Press a red LED fully into the hole of a “brick 2x1 with hole” and place it on a red “brick 2x2”.
Observe the same comments as above.
Q1. Why is the light sensor assembly identical to the light source assembly?
3.3.2 Optimize the Light Sensor
Connect the differential voltmeter and the current sensor to channels 1 and 2 on the SensorDAQ.
Download the Colorimetry VI from e-Learning and open it in LabVIEW. Open the Block Diagram and
take a few minutes to figure out what the VI does.
Before connecting anything to the voltmeter or current sensor start up the VI and once the readings have
stabilized, push the “Tare” button.
Q2. What happened when you pushed the Tare button? How did the readings change?
Now, connect the light sensor to the differential voltmeter (circuit diagram 2) and observe the background
reading.
Circuit Diagram 2
Q3. What is the background under ambient light?
The optimum background reading is 0 mV. Cover the light sensor with a black cloth or garbage bag.
Depending on the ambient light conditions, different degrees of covering may be needed. Try folding the
garbage bag to provide more layers of protection. Cover the windows to the room, if possible.
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Colorimetry |7
Test different orientations of the colorimeter (with the light sensor pointing toward or away from the
window, for example.) Aim for the lowest background reading you can achieve without disturbing the
arrangement too much.
Q4. What is the lowest background voltage you were able to achieve?
Q5. What conditions resulted in the lowest background voltage?
3.3.3 Turn on the Light Source
1. Add the current sensor to the circuit powering the light source. Leave the voltmeter to read the
voltage produced by the light sensor.
2. Turn on the light source by closing the switch in the circuit.
3. Pipette 3 mL of DI water into a clean cuvette. This is your reference cuvette.
4. Place the reference cuvette into the cuvette holder block.
5. Note the voltage from the light sensor.
The intensity of the light source will vary for the first 5-10 minutes while the LED warms up. While you
wait for the LED to stabilize you can begin the serial dilutions of your food color solutions.
3.3.4 Serial Dilution in Cuvettes
Cuvettes must always be treated with the care accorded to optical instruments. Do not touch the flat clear
surfaces with your fingers to avoid leaving marks that will interfere with the light path. Handle them only
by holding them at their corners, or on the opaque surfaces on some cuvettes.
Use a sharpie to label your five cuvettes A – E, near the top of the cuvette, away from the light path.
Follow the instructions below to create a set of 4 dilutions of the stock solution.
Cuvette
Instructions
A
Pipette 2 mL of the stock solution into cuvette A. Replace cuvette A into the cuvette holder.
B
Pipette 1000 µL of the stock solution into cuvette B. Pipette 2000 µL DI water into the
cuvette. Mix by gently pipetting up and down. Avoid making bubbles, which will interfere
with the light path. Replace cuvette B into the cuvette holder.
C
Pipette 1000 µL of the solution from cuvette B into cuvette C. Pipette 2000 µL DI water into
the cuvette. Mix as above. Replace cuvette C into the cuvette holder.
D
Pipette 1000 µL of the solution from cuvette C into cuvette D. Pipette 2000 µL DI water into
the cuvette. Mix as above. Replace cuvette D into the cuvette holder.
E
Pipette 1000 µL of the solution from cuvette D into cuvette E. Pipette 2000 µL DI water into
the cuvette. Mix as above. Replace cuvette E into the cuvette holder.
Prepare a table in Excel to calculate the dilution factor of each dilution. The food color is from the
grocery store, and does not come with a concentration. In a future lab, you will measure the molar
concentration of FD&C #1 in the stock solution. Until then, assume the stock solution has concentration
of of 1.00 AU (arbitrary units).
3.3.5 Measure the Transmittance as a Function of Concentration at 626 nm
Check to confirm that your light source has stabilized and that your reference cuvette is still in the cuvette
holder block.
You will measure the transmittance in the units of voltage produced by your light sensor.
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Prepare a table in your lab notebook to record the transmittance (in mV) of the reference cuvette and each
dye cuvette. If your light source is still changing, you may need to record a reference reading for each dye
cuvette.
3.3.6 Analysis of Transmittance Measurements
Use your Excel workbook to calculate the percent transmittance of each measurement using the relation
%𝑇 =
𝑉𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛
∙ 100%
𝑉𝑟𝑒𝑓𝑒𝑟𝑒𝑛𝑐𝑒
and to plot a graph of transmittance as a function of concentration (in arbitrary units).
Q6. Use the Snipping Tool to snip a copy of your plot and paste it here.
Q7. Is the relationship between transmittance and concentration linear? If not, what is the relationship?
Note that you are defining transmittance as
𝑇=
𝑉𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛
𝑉𝑟𝑒𝑓𝑒𝑟𝑒𝑛𝑐𝑒
From the transmittance results, calculate the absorbance of each measurement
𝐴 = −𝑙𝑜𝑔𝑇 = −𝑙𝑜𝑔 (
𝑉𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛
)
𝑉𝑟𝑒𝑓𝑒𝑟𝑒𝑛𝑐𝑒
Plot a graph of absorbance as a function of concentration (in arbitrary units).
Q8. Use the Snipping Tool to snip a copy of your plot and paste it here.
Q9. Is there a linear relationship between absorbance and concentration? If not, what is the relationship?
3.4
POST-LAB ASSIGNMENT
Work with your extended group to submit a one-page abstract in class, describing the experiment you just
completed. Include your figure in your abstract.
For details, refer to the Abstract Writing Guidelines posted in the Student Resources folder on eLearning.
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