Understanding Colorimetry
Part of:
Inquiry Science with Dartmouth
Developed by Ramsa Chaves-Ulloa (with Debbie
Groveman and Brad Taylor)
Overview
Colorimetry is a method of determining the concentration of a substance in a solution. This method has
wide uses in science including determining the concentration of hemoglobin in blood, of chemicals such
as chlorine, cyanide and mercury in water, of glucose and chlorophyll in plants, and of nutrients in the
soil. This lesson teaches students to build a standard curve to determine the relationship between the
light absorbed by a substance and its concentration in a solution.
Science Standards
Science Process Skills
S:SPS1:8:1.1 Use appropriate tools to accurately collect and record both qualitative and quantitative data gathered
through observations (e.g., temperature probes, electronic balances, spring scales, microscopes, stop watches).
S:SPS1:8:1.7 Ask questions about relationships between and among observable variables.
S:SPS1:8:3.1 Use appropriate laboratory techniques to carry out student- or teacher-developed procedures or
experiments.
S:SPS1:8:3.2 Use appropriate tools to gather data as part of an investigation (e.g., ruler, meter stick, thermometer,
spring scale, graduated cylinder, calipers, balance, probes, microscopes).
S:SPS1:8:3.3 Follow the teacher’s instructions in performing experiments, following all appropriate safety rules and
procedures.
S:SPS1:8:4.1 Use appropriate tools (including computer hardware and software) to collect, organize, represent,
analyze and explain data.
S:SPS1:8:4.3 Draw appropriate conclusions regarding the scientific question under investigation, based on the data
collected.
S:SPS3:8:1.1 Work effectively within a cooperative group setting, accepting and executing assigned roles and
responsibilities.
S:SPS3:8:1.2 Work collectively within a group toward a common goal.
S:SPS3:8:1.3 Demonstrate respect of one another’s abilities and contributions to the group.
Life Science
S:LS5:8:2.1 Recognize and provide examples of how technology has enhanced the study of life sciences, as in the
development of advanced diagnosing equipment improving medicine.
Physical science
S:PS4:8:2.1 Demonstrate appropriate use of tools, such as rulers, calculators, balances, and graduated cylinders to
measure and calculate volume and mass.
Focus Question
What is Colorimetry? How has this technique enhanced the study of science? What is light absorbance?
What is a standard curve?
Objectives
Through this lesson, students will:
 Learn about the colorimetric method and its uses in science
 Learn about the relationship between light absorbance and the concentration of a substance in a
solution
 Learn how to build standard curves
 Use appropriate tools (graduate cylinders and rulers) to accurately collect and record quantitative
data to build standard curves
 Use computer software (Microsoft excel) to collect, organize, represent data to build standard
curves
 Learn the predictive value of standard curves
Background
Colorimetry works because of light absorbance. A substance in a solution absorbs light, which decreases
the amount of light that passes through the solution. The amount of light absorbed by a solution is related
to the concentration of a substance in that solution. We are going to make a standard curve of milk in
water.
Vocabulary
Colorimetry: The science and technology used to quantify and describe physical color as
perceived by humans
Light absorbance: the measure of the attenuation of light. Attenuation can be caused by absorption, but
also reflection, scattering, and other physical processes.
Standard curve: a type of graph used as a quantitative research technique. Multiple samples with known
properties are measured and graphed, which then allows the same properties to be determined for
unknown samples by interpolation on the graph. The samples with known properties are the standards,
and the graph is the standard curve.
Materials (for 20 people working in groups of two)
10 100 ml clear plastic containers
1500 ml of water
1 tape roll
10 50 ml graduated cylinders
250 ml of whole milk
10 droppers
10 rulers
10 markers
10 laser pointers
** In this lab the students will be using a laser pointer. It is important to point out that lasers are
NOT toys and can severely damage people’s eyes.
Preparation
Collect materials.
Procedure
1) Research: Talk about how colorimetry is relevant for your research. Introduce the concept of light
absorbance and its relationship with the concentration of a substance in a solution. You can also
expand by talking about spectrophotometry. Brief power point presentation <5 minutes.
2) Hook: Show images of the use of colorimetry in science. For example in medicine (e.g., test the
concentration of hemoglobin in blood), environmental sciences (e.g., to test mercury
concentrations in streams and lakes), and biology (e.g., test glucose concentration in plants). <5
minutes
3) Background: discuss the concepts of light absorbance and its relationship with concentration.
Discuss the concept of building a standard curve.
4) Experiment: Form pairs and give each group one plastic
container, a graduate cylinder, a dropper, a ruler, a marker and a
laser point and a data sheet. Ask each pair to find a spot
approximately 5 feet away from a wall where they can set up a
station. Students will need to be able to shine a light onto the
wall or flat surface. Ask the students to tape the “data sheet” onto
the wall or surface. The students will add 50 ml water to the
containers, and shine the laser pointer through the container so
that they can see a red spot on the data sheet. The laser pointer should aim to get a spot on the
line on the data sheet corresponding to 0 (zero) drops of milk. The students should use a pencil
and circle the red spot. The spot will be a bit fuzzy, but students should use their best judgment
to decide what the diameter should be. Students should use a ruler to measure and record the
diameter of the spot. Students should record the data in a data table.
5) Extend the experiment: Students should add ten drops of milk to the container. Mix to evenly
distribute the milk. They should use exactly the same set up and shine the laser through the
container, circle the spot and measure the diameter. The red spot should be fainter, and the length
should be smaller. Students should record the data in a data table.
6) Students should add 10 more drops (a total of twenty) to the container. They should follow the
same procedure and record the diameter of the red spot again. Once again, the red spot should be
fainter, and the length should be even smaller. Students should record the data in the data table.
7) Plot: students should enter their data in Microsoft excel and build the standard curve by graphing
the data and adding a line through the points. Remind the students to label each axis and add a
title. Ask the students to identify the independent and the dependent variables.
Assessment
1) Based on the standard curve built by the students test if the standard curve works. Ask the
students to predict the length of the line if you were to have 15 drops of milk.
2) Ask students to rinse the container and make a new sample (50 ml water + 15 drops of milk) to
carefully test their prediction. Record the laser spot length, exactly as before. EVERYTHING
MUST BE EXACTLY THE SAME FOR THE STANDARD CURVE TO WORK!! Make sure
the students have the container the same distance from the surface, the same pointer and observer,
etc. Ask students to answer if the predicted value was the same as the experimental measurement,
and to explain their result.
3) Ask the students to rinse the container and build a mystery sample. Each group should label their
containers in a unique way. Then ask students to exchange their mystery sample with another
group. Ask the groups to measure the concentration of milk in their mystery samples.
4) Solve the mystery! The students should use their standard curve to determine how many drops of
milk were in their mystery containers.
5) Verify the student’s calculations.
Extensions
Use colorimetry to measure phosphorus in streamwater. You could use a spectrophotometer to do this and
explain the students that spectrophotometers use the concept of colorimetry but using the IR spectrum
instead of visible light.
Understanding Colorimetry
Group notes and results
Experiment
Data table
# of drops
0
Length (cm)
10
20
a. what is the independent variable? ____________________________________
b. what is the dependent variable? _____________________________________
Assessment
Based on your standard curve predict the length of the line if you were to have 15 drops of milk _________________
measured value for 15 drops of milk ____________________________
Was your predicted value the same as your experimental measurement? ____________
Briefly explain why or why not ? __________________________________________
Mystery
solution
Length (cm)
Experimental
value drops
# of Drops
0
5
10
Mystery Vial #
A
B