ChemDetectives
Chromatography: Separating Good from Bad
Or
How to Find the Source of Insecticide
Contamination Using Chemistry
Erin “Dr. E” Nyren, PhD, MBA
HQC Biosciences, Inc.
Age level: 13-16 years of age
Learning Objectives:
By the end of this lesson, students will be able to:
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Describe the concept of solubility
Define and describe the words analyte, chromatography, mobile phase, stationary phase,
and retention factor
Explain how differences in solubility may be used to separate chemical components
(i.e.—individual compounds that are part of a complex mixture) in an analyte.
Give other examples of differences that may exist between chemical components within
analytes which may be used to separate them
Explain what a standard is, and the importance of using standards in a chromatography
experiment
Perform thin layer chromatography with good separation of individual chemical
components within the analyte(s).
Quantify results of thin layer chromatography by measuring retention factor (Rf) values
of the different chemical components.
Describe reasons why one would wish to separate chemical compounds in an analyte.
Prepare a written report describing the procedures and outcomes of the experiment.
Key Concepts:
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Some materials are composed of multiple chemical components that may have different
properties (solubility, size, charge).
We can separate chemical components in a solution based on solubility, size, and/or
charge.
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What’s Worrying the Doctors in Farmington?
Monday morning, 8:00 am, the door shuts softly behind you as you enter your office. As the
lead chemist for the state lab in Farmington, you have a busy day ahead of you—there is already
a message on your answering machine (this is unusual so early on a Monday morning). The
message is from Dan at the local police department, and he says it’s urgent that you return his
call; he has a large volume of samples for your lab to test, and they must be given high priority.
This is a serious concern—you are already very busy, and wonder what could possibly make the
samples so urgent.
When you return Dan’s call, he explains that a doctor from Children’s Medical Center contacted
him about a month ago with some serious concerns. He has been carefully monitoring the
number of birth defects in babies born in Farmington over the last ten years, and in the past two
years he has noticed that the number has jumped from one child out of 33 (the typical national
average1) to one child out of 10. None of the families concerned have had any known contact
with any chemicals known to cause birth defects, such as drugs or alcohol, so there is no
immediate explanation for the increase. This is not only devastating to the families involved; it
is frightening the entire community! You ask Dan if he has any thoughts as to what could be
causing such a rise in birth defects in Farmington. Dan says he thinks he does.
Recently there have been rumors circulating around Farmington about one of the local farmers
hiring an obscure crop duster to spray his crops with insecticide. The pilot claimed to be a fully
registered and licensed crop dusting business, and was offering crop dusting services at a fraction
of his competitors’ prices, but he was later investigated for not having proper documentation.
When asked what he uses on his clients’ crops, he claimed he used INV-80, a common
insecticide used by most of the farmers in the area. However, operators of other crop dusting
businesses in the area all suspect he is actually using QU-10, an inexpensive and extremely
effective insecticide that has been banned in the US. The name QU-10 is very familiar to you;
you were part of a research team that studied the long term effects of QU-10, and you
immediately recall that your research team concluded QU-10 causes birth defects—the same
type of birth defects currently on the rise in Farmington!
Dan goes on to say that when the suspected crop duster was brought in for questioning, he firmly
denied ever using QU-10, but thought that some of his competitors might. Because the use of
QU-10 is suspected, and because it could put the whole town of Farmington at risk, Dan was
authorized to collect several soil samples from every farm in the area—60 samples in all. He
says you should expect to receive them today.
A few hours later you receive a large box containing 60 samples of local soil. You soak each
one in a mixture of alcohol and water to extract any pesticides, and remove any leftover rocks
and large pieces. Now you have 60 samples to test for the presence of QU-10. Knowing that
1
According to the Centers for Disease Control and Prevention, www.cdc.gov
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QU-10 has no color, no odor, and is nearly indistinguishable from other insecticides used in the
area, you know you will need to use chemistry to find out if someone is using it, and potentially
poisoning the people of Farmington.
How Do We Use Chemistry to Catch the Culprit?
While we know that QU-10 looks just like any other safe insecticide, we also know that it is
chemically different from most other insecticides used in Farmington. Because its properties are
different from other insecticides, we can use some simple chemical analysis techniques to see
which soil samples (if any) contain QU-10.
Chemical Compounds Can Be Separated Based on Their Chemical or Physical Differences
You now have alcohol-based extracts of soils from all the surrounding farms to investigate.
These extracts are your analytes, the materials you are going to analyze. To separate the
different chemical compounds in these analytes, we need to think about how they might be
different from each other. Some differences that may exist between the different compounds are:
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Differences in solubility—that is, their tendency to be dissolved in water or oil. This
depends on whether the compounds are more hydrophilic (water loving) or more
hydrophobic (water hating). Even if the compounds dissolve in water, one may still be
more hydrophilic than another (see Fig. 1).
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Differences in size—sometimes the compounds we are interested in are distinct in size or
shape from the other compounds, helping us separate them even if they have identical
solubility (See Fig. 2).
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Differences in charge—two compounds may have equal solubility in water and equal
size and shape, but they may still be separated if one has a positive charge and one has a
negative charge, or even if one has a greater charge than the other (say, one chemical
compound carries a +2 charge, while the other carries a +1 charge).
When we separate chemical compounds based on these differences, we use a technique called
chromatography.
What is Chromatography, and How Does it Work?
Chromatography is the technique we use to separate complex mixtures into their different
components. While there are several different types of chromatography, they all have a few
things in common:
1. A Mobile Phase: This is the liquid that the analyte is dissolved in, such as water or
alcohol, and which will move during the separation, carrying the different compounds in
the analyte with it.
2. A Stationary Phase: This is the solid material that the mobile phase moves through, and
which the analyte will also interact with as it moves, carried by the mobile phase.
As the analyte passes over the stationary phase, some of the chemical components will interact
more strongly with the stationary phase than others. The slight differences in solubility will
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cause each component to be slightly more attracted to the stationary phase (more hydrophilic) or
the mobile phase (more hydrophobic), and this slight difference in attraction for one or the other
phase will allow these chemical components to separate from each other. The more attracted a
chemical component is to the stationary phase, the slower it will move during the
chromatography; the more attracted a component is to the mobile phase, the faster it will move
(See Fig. 4).
Depending on the type of chromatography used, we can separate chemical components based on
their size or charge in a similar way.
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In the following activities, you will use thin layer chromatography to separate your soil samples
into different components.
Use Chromatography to Find Who is Using QU-10
We can now use techniques like those described above to find out which (if any) of the farmers
around Farmington are using dangerous QU-10 on their fields. We know that we can’t tell the
difference between QU-10 and other insecticides that are safe to use by looking at them or
smelling them, but we know that the chemical structure of QU-10 makes its solubility in water
different from most other insecticides that are in use. This means we can separate QU-10 from
other insecticides using chromatography! For this we will use a very common and very useful
form of simple chromatography called thin layer chromatography.
Thin layer chromatography (or TLC) is a type of chromatography used to separate mixtures of
simple chemical compounds. Our stationary phase is a thin layer of silica supported by a piece
of glass or plastic. This is called a TLC plate. Our mobile phase for this experiment will be a
mixture of 70% isopropyl alcohol (that is, rubbing alcohol) and 30% water.
For our experiment, we will run four samples for each thin layer chromatography strip: two
samples of soil we need to test, and two standards, one for QU-10 and one for a very commonly
used safe insecticide, INV-80. A standard is a known sample (that is, a known material) that we
use for comparison. By comparing our soil samples to the known insecticide standards, we can
tell if any of the samples contain dangerous QU-10, or just INV-80, which we know is safe to
use.
What you will need:
1.
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Latex or other protective gloves
TLC plates coated with silica
70% Isopropyl alcohol for use as mobile solvent
Development chamber
Samples of alcohol-based extractions of soil
Samples of insecticides to use as standards
Capillary tubes (These are small, plastic tubes that are hollow in the middle. You will
use these to transfer your sample from the vials to your TLC plate.)
8. A ruler
9. A pencil
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Time you will need: 30 minutes
1. Put on your protective gloves before handling your samples!
2. Using your ruler and pencil, draw a thin line two centimeters
from the bottom of your TLC plate (see Fig. 7). Be sure not to
press too hard on the silica coating! (Remember, the silica
coating is hydrophilic, which makes it a good stationary phase
for our chromatography!)
3. Use the capillary tubes to place a spot of the standars and your sample on this pencil line
(see Fig. 8). BE SURE TO USE A FRESH CAPILLARY TUBE FOR EACH SAMPLE
AND STANDARD!
4. Measure ¼ cup of the isopropyl alcohol, and pour it into the
development chamber. (Remember, the isopropyl alcohol is
slightly more hydrophobic, making it a good mobile phase for our
chromatography!)
5. Slowly lower the TLC plate with the samples on it into the
development chamber. Lean the plate against the side of the
chamber, and make sure it is standing upright (see Fig. 9).
MAKE SURE THE SAMPLE SPOTS ARE STILL ABOVE
THE RUBBING ALCOHOL!
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6. Very carefully put the lid on the development chamber, without letting the rubbing
alcohol slosh around. The lid does not need to be very tight.
7. Watch as the isopropyl alcohol starts to rise up the TLC plate! It
will meet the sample, which will also start to move up the TLC
plate, although you won’t be able to see it move. Let the plate sit
in the rubbing alcohol this way for at least 20 minutes, or until
the rubbing alcohol has traveled about 2-4 centimeters from the
top of the TLC plate (see Fig. 10).
8. Carefully remove the jar lid, and pull out the TLC plate. Lay it
on the table or counter, and use your pencil to trace the line where the alcohol stopped
moving.
9. Allow the plate to dry for about 5-10 minutes.
10. Turn off the room lights, and use a UV light to visualize the chemical components as
fluorescent spots on the plate. Use a pencil to carefully trace around the edges of the
spots.
Analyze Your Results
In order to look at your thin layer chromatography results objectively (that is, to remove all bias),
and to understand how they compare to the results of others who have done the same experiment,
it is useful to assign a numerical value to describe how far along the paper each chemical
component traveled. These values are called retention factors, or Rf values, and they are
measured by calculating the ratio of the distance traveled by the chemical component to the
distance traveled by the mobile phase, rounded to two decimal places. Here is an example:
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Suppose Figure 11 below represents a chromatography plate with a single sample when the
experiment is finished, as seen under a UV light.
We can see that the sample has separated into three different chemical components, which are
visible as separate spots under a UV lamp. We also measured the distance from the first pencil
line where the samples were first spotted and the line where the solvent stopped moving when
the chromatography was stopped. This distance is 8 centimeters (cm). The three chemical
components have traveled 2 cm, 4 cm, and 7 cm, as measured from the line where the sample
started to the middle of the visible spot on the plate.
We now must calculate the Rf values of the different chemical components of the sample.
Component number 1 has traveled 2 cm, therefore the Rf value for this component is:
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑒𝑑 𝑏𝑦 𝑐𝑜𝑚𝑝𝑜𝑛𝑒𝑛𝑡 1 2 𝑐𝑚
=
= 𝟎. 𝟐𝟓
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑒𝑑 𝑏𝑦 𝑚𝑜𝑏𝑖𝑙𝑒 𝑝ℎ𝑎𝑠𝑒 8 𝑐𝑚
Component number 2 has traveled 4 cm, therefore the Rf value for this component is:
4 𝑐𝑚
= 𝟎. 𝟓
8 𝑐𝑚
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Component number 3 has traveled 7 cm, therefore the Rf value for this component is:
7 𝑐𝑚
= 𝟎. 𝟖𝟖
8 𝑐𝑚
We can now use these values for our chemical components, and describe very clearly and
precisely the results of our thin layer chromatography.
Once you have measured the Rf values of both the spots from the QU-10 and INV-80 standards
and the value or values of the spots from your samples of soil, compare the Rf value of each
sample to that of the QU-10 and INV-80. Are the numbers the same or very close? Write the
numbers below:
QU-10 standard Rf: ____________________
INV-80 standard Rf: ___________________
Sample Rf: _______________________
Based on these results, do you believe that your sample of soil contains dangerous QU-10? Why
or why not?
Based on these results, do you think QU-10 is more soluble in water (more hydrophilic), or more
soluble in alcohol (less hydrophilic)? Why or why not?
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Final Report:
Prepare a report discussing the results of your paper chromatography. Use the report format and
instructions on page 15.
Why Else Would We Use Thin Layer Chromatography?
Now that you know how to perform thin layer chromatography, think like a chemist! Why else
would you need to use such a technique? Here are some typical uses for thin layer
chromatography:
Find out if a compound or compounds of interest are in a sample by comparing them to a
standard. This is exactly what you have just done: you analyzed your soil sample to see if it
contained QU-10 or INV-80, and used standards to compare to your unknown.
Separate the compounds in a complex sample in order to test them individually. Imagine
you are studying a plant that appears to have fungicidal properties (e.g. the leaves of this plant do
not become infected with fungi, as the leaves of other plants do). You would like to know if it is
a single compound in the leaves of this plant that make it resistant to fungal growth, so you
prepare an extract of the plant leaves and perform TLC on this extract. Now you may use your
finished TLC plate for some further experiments. For example, you could now spray the TLC
plate with the separated compounds on it with some of the fungus that the plant is resistant to,
and see if there is a specific place on the plate where the fungus won’t grow (see Figure 12)!
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Monitor a chemical reaction to determine if it is finished. Suppose you are trying to develop
a new insecticide. You are performing a chemical reaction by adding compound ‘A’ to
compound ‘B’, and the product of the reaction is compound ‘C’. As a chemist would write, this
chemical reaction looks like this:
The final product of this reaction is colorless, so we do not know when the reaction is complete
simply by observing it (although we can see compounds A, B and C under a UV light). We can
use TLC to test the reaction mixture and compare it to the starting compounds to check if
compounds A and B are reacting to form compound C (see Figure 13).
Additional Sources:
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“Chromatography”. Wikipedia. www.wikipedia.org/wiki/Chromatography
“Thin Layer Chromatography (TLC)”. Organic Chemistry at CU Boulder.
http://orgchem.colorado.edu/Technique/Procedures/TLC/TLC.html
“Thin Layer Chromatography”. UC Davis Chemwiki.
http://chemwiki.ucdavis.edu/Reference/Lab_Techniques/Thin_Layer_Chromatography
 Lundanes, E. “Chromatography: Basic Principles, Sample Preparations and Related
Methods” (2013). Wiley-VCH, Weinheim, Germany.
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Written Report Format:
Your reports should include the following sections and structure:
Title Page:
Project Title
Your Name
Course Name
Date
Introduction: In this section you describe the purpose of your experiment, and provide
some brief background about the importance of your experiment. You should provide a brief
description of the procedures you will be using, and why you have chosen these methods for
this experiment.
Procedures: Describe in detail the materials you have used, such as the TLC Plates, the
capillary tubes, the mobile phase, and the container you used for the thin layer
chromatography. Describe the method you followed during your experiment step by step,
including the way in which you calculated the Rf values. Provide as much detail as possible,
so that anyone else reading your procedures could perform the same steps as you have, and
get the same results.
Results: In this section you provide the results of your experiment objectively—that is, you
describe exactly what happened. In this section you should also provide a table of your
calculated Rf values. Do not discuss why you believe you got these results or interpret them;
you will do this in the next section.
Discussion: Here is where you discuss why you got the results you did, and discuss what
you believe could have been done differently. For your chromatography experiment, here is
where you explain which of the insecticides your soil contains (if any), if you had any
difficulties with the experiment, and if you believe the results could have been different
under different circumstances.
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Conclusion: Summarize your results and their interpretation. This section should be brief—
four or five sentences—but should concisely explain what happened and why.
References: In order to gather information about the procedures you used and describe the
importance of your experiment, you should use external sources, such as text books and
journal papers. List these here in order of their use, using consistent citation format. CSE
style is suggested. For more information about CSE style, please visit the Washington State
University Library website at http://www.wsulibs.wsu.edu/quickguides/cse.
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Glossary:
Analyte: Material generally made up of multiple chemical components which is prepared for
analysis.
Standard: A known sample which is analyzed the same way as unknown samples during an
experiment to use as a comparison.
Mobile Phase: The liquid in which the analyte is dissolved, and which moves along the
stationary phase carrying the analyte.
Retention Factor: The ratio of the distance traveled by the chemical component to the distance
traveled by the mobile phase during paper chromatography.
Solubility: A material’s tendency to be dissolved in one substance over another, such as water
or oil.
Stationary Phase: The solid material that the mobile phase moves through, and which the
analyte will also interact with as it moves, carried by the mobile phase.
Thin Layer Chromatography: A method of chromatography used to separate mixtures of
simple compounds. This technique uses a thin layer of some hydrophilic material, usually silica,
aluminum oxide, or cellulose, on a glass or plastic support.
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Appendix A: A model for the concept of chromatography
Materials:
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A fine sieve
A large cloth or paper towel, approximately 12” x 12”
A bowl large enough to hold 2 cups of water
¼ cup of pea gravel
¼ cup of fine sand
2 cups water
Preparation (See Fig. A):
1. Mix together the pea gravel and the fine sand. Set aside.
2. Place the paper towel in the bottom of the bowl, the sides hanging over the edges, and the
sieve on top of the paper towel.
3. Have the two cups of water ready in a separate container off to the side.
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Demonstration:
The sieve, paper towel, and bowl represent the
stationary phase of our chromatography. The water
represents our mobile phase. The mixed pea gravel and
sand represent our sample.
Show the students the sample. Ask what is the most
obvious difference between the two compounds in the
sample? Clearly, they are different in size. We can use
“chromatography” to separate these two compounds in
our sample.
Pour the pea gravel and sand “sample” into the
sieve/paper towel/bowl “stationary phase”, and pour the
water “mobile phase” over them (see Fig. B). The pea
gravel should stay in the sieve; while the sand is caught
by the paper towel (see Fig. C).
This is similar to how chromatography separates the compounds in our sample: the mobile
phase carries the sample through the stationary phase, and the different compounds interact
differently with the stationary phase versus the mobile phase. This causes the different
compounds to separate based on the differences in their properties (like size, in this case, or
solubility in the case of our thin layer chromatography).
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