Chromatography:

How Can We Use Chromatography to Separate Plant Pigments?

▶ For this experiment, you need to bring a pencil and a leaf sample (a green leaf of any type, but not dry and
crumbly) to lab.

Introduction

Most substances found in nature are composed of complex mixtures of compounds. In order to obtain a better un-
derstanding of these substances, it is often useful to separate them into their various components. The separation
procedures rely on the differences in chemical and physical properties between the various components of the mix-
ture. The aim of this experiment is to introduce you to one of the more common and powerful separation techniques,
chromatography.

In 1906, a Russian botanist, Mikhail Tsvet, devised a new and ingenious method for the
separation of plant pigments. By pouring an extract of leaf material through a vertical
glass tube packed with powdered chalk, he observed that the extract separated into several
distinct, colored bands, as shown in Figure PP.1. He named his process chromatography.
He was further able to isolate the materials from each band by pushing the chalk out of the
tube, cutting between the zones, and extracting each band with alcohol. Using this column
chromatography technique, he separated a complex mixture into its components.

Chromatography has wide applications in chemistry. When chromatography is used to

separate colored substances, the separated components can be identified visually. When
colorless substances, such as amino acids, are separated by chromatography, a dye or UV
light is used to locate the separated components on the chromatogram. The original col-
Figure PP.1:

umn chromatography technique has led to many different separation methods such as pa- Reproduction of
Mikhail Tsvet’s
per chromatography, thin-layer chromatography (TLC), and gas chromatography (GC). Al-
separation of plant
though modern chromatographic methods employ various techniques and equipment, they
pigments using an
share a common characteristic with the original column method: they all separate mixtures extract of spinach.
into individual components by the relative solubility of the components between a sta-
tionary phase (the composition of the column or substrate) and a mobile (moving) phase (the solvent used in the
separation). The variation of attraction is due to different intermolecular interactions between the compounds in the
mixture and the stationary and mobile phases.

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 How Can We Use Chromatography to Separate Plant Pigments?

Thin Layer Chromatography

In this experiment, you will become familiar with separations by thin-layer chromatography, TLC. The stationary
phase in TLC is a thin layer of silicon dioxide (silica), or sometimes aluminum dioxide (alumina), coated onto a glass,
metal, or rigid plastic sheet. The outer surface of the silica is covered in –OH groups, as shown in Figure PP.2. The
surface of the silica is very polar and will interact with polar components in the mixture. In addition, the –OH groups
are available to hydrogen bond with hydrogen-bonding components in the sample. For the mobile phase, a suitable
solvent or solvent mixture is chosen based on the components to be separated.

Figure PP.2: Partial image of the surface of silicon dioxide (silica). Note the –OH groups on
the surface.

When a sample is applied, or spotted, near the bottom edge of the TLC plate and the edge of the plate is placed in
a solvent, the solvent (mobile phase) rises by capillary action and flows up the plate. When the leading edge of the
mobile phase (the solvent front) reaches the sample, the sample components dissolve and they move up the plate
with the solvent. How fast the compounds move up the plate depends on two factors:

• The solubility of the compound in the solvent. This is dependent on how strong the intermolecular forces
are between the solvent and the compound.
• How attracted the compound is to the silica surface of the plate. This is dependent on how strong the
intermolecular forces are between the silica and the compound.

Most compounds, whether ionic or molecular in nature, are somewhat attracted to both stationary and mobile phases.
Those compounds that are more attracted to the mobile phase will spend more “time” in the mobile phase, and will
travel further up the TLC plate. Those compounds that are more attracted to the stationary phase will spend more
“time” in the stationary phase, and will not travel as far up the TLC plate. This relative attraction of the compounds
to either the mobile phase or the stationary phase will cause the components of a mixture to move up the plate at
different rates and separate, producing a chromatogram. We can estimate the relative attraction of different species
by looking at the polarity of the mobile and stationary phases and the relative polarity of the compounds. If the
mobile phase is non-polar, compounds that are less polar will want to spend more time with the mobile phase and

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 How Can We Use Chromatography to Separate Plant Pigments?

travel further up the TLC plate. Polar compounds will want to spend more time with the polar stationary phase and
not travel as far up the TLC plate.

The steps involved in separating a mixture using TLC are outlined in Figure PP.3.

Figure PP.3: A separation process using thin layer chromatography.

a. A TLC chromatogram is “loaded” by placing small spots of the mixture to be separated on a defined line
(baseline).
b. The chromatogram is placed into a chamber containing the solvent. The TLC plate is porous, so it will
begin to absorb the solvent. The baseline loaded with the mixture is initially above the solvent.
c. A lid ensures that the atmosphere in the chamber is saturated with solvent. As the solvent rises, it carries
the components of the mixture with it. Colored compounds will appear on the chromatogram as spots.
d. As the solvent moves up the plate, the mixture separates into a fast-moving spot and a slower moving spot
(or spots).
e. When the solvent front is close to the top of the plate, the plate is removed from the chamber and the
solvent front is marked for reference.
f. Once separated, the components can be identified by comparison to the scientific literature or by reference
against a known standard.

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Retention Factor (R𝑓 )

We cannot directly identify components separated by TLC. Instead, we need to compare the distance the spots of the
sample traveled to the spots of a standard mixture. This comparison is done by calculating the retention factor, R𝑓 ,
of each component. An R𝑓 value is simply a ratio of the distance the compound traveled from the baseline divided
by the distance the solvent front moved from the baseline. An R𝑓 should have a value between 0 and 1.

Distance Compound Moved

R𝑓 = (Equation PP.1)
Distance Solvent Moved

Figure PP.4: Example of an R𝑓 calculation.

In the examples shown in Figure PP.3 and Figure PP.4, the R𝑓 is = 5/6 (= 0.83) for the fast-moving spot and 2/6
(= 0.33) for the slower spot. The R𝑓 of the faster spot is larger, meaning that it has moved farther along the TLC plate
than the slower spot. When a non-polar solvent is used for the separation, it can be concluded that the compound)
in the fast-moving spot (red) is less polar (more non-polar) than those is the slow-moving spot (blue).

R𝑓 values are constant for a compound under the same experimental condi-
tions. This is beneficial for TLC because we can simultaneously separate and
analyze a standard mixture and our samples. If we perform a TLC separation
and get the example plate shown in Figure PP.5 below, the only conclusions
we can accurately draw from the experiment is that we have compounds A
and C in our sample. We also know we don’t have compound B. We don’t
know anything about compound D because it’s not present in the standard.

In order to determine the components of the plant pigments analyzed in

this lab, you will prepare a reference standard for comparison. While the
R𝑓 values on your chromatogram may differ slightly, the relative location Figure PP.5: Example TLC Plate

and color of each of the components should remain constant and can be used to determine if any of those components
are present in your sample(s).

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 How Can We Use Chromatography to Separate Plant Pigments?

Using TLC to Separate Pigments found in Plants

TLC is ideally suited for separating biological mixtures, such as color pigments in plants, because it is simple and
quick to perform compared to other chromatography methods.

Other advantages of separation by TLC are:

1. Only a small amount of sample is required.

2. The separated compounds can be visualized either by the naked eye or by UV light.
3. The sample mixture can contain chemical compounds beyond those of interest without adversely affecting
the separation of the compounds of interest.
4. The separation is based on more than one property of the compounds, making it easier to separate similar
but not identical molecules as seen in the separation of chlorophyll a and chlorophyll b (Figure PP.6).

The individual pigments found in the leaves of plants can be separated and analyzed by TLC. The components can
then be identified by comparison to a known standard. The two main types of pigments found in plants are chloro-
phylls and carotenoids. In this experiment, you will attempt to separate and identify the chlorophylls and carotenoids
in leaf samples by comparison with a spinach standard.

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 How Can We Use Chromatography to Separate Plant Pigments?

Chlorophyll Pigments

Chlorophyll is a green pigment that comes in three forms: a, b and c. The three forms are found in a variety of
different plants and are essential for photosynthesis. Spinach primarily contains chlorophylls a and b, the structures
of which are shown below. The structures have a subtle but important difference (highlighted in red in Figure PP.6),
which will enable them to be separated by TLC.

Chlorophyll a usually appears as a blue-green spot with a retention factor ranging from 0.61–0.63 (in 4 mL of acetone
and 6 mL of hexane). Chlorophyll b usually appears as a green spot with a retention factor ranging from 0.59–0.61
(in 4 mL of acetone and 6 mL of hexane).

Figure PP.6: Structures of Chlorophyll a and b.

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 How Can We Use Chromatography to Separate Plant Pigments?

Carotenoid Pigments

Carotenoids are red, orange or yellow pigments and include two subtypes: xanthophylls and carotenes. These pig-
ments are responsible for giving carrots, tomatoes and other fruits and vegetables their red, orange or yellow colors.
The difference between lutein (a xanthophyll) and beta-carotene is highlighted in red in Figure PP.7.

Xanthophylls usually appear as one or two yellow spots with retention factors ranging from 0.55–0.58 (in 4 mL of
acetone and 6 mL of hexane).

Beta-carotene usually appears as a yellow-orange spot with a retention factor ranging from 0.93–0.95 (in 4 mL of
acetone and 6 mL of hexane).

Figure PP.7: Structures of carotenoid pigments lutein (a xanthophyll) and beta-carotene.

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Goal

The purpose of this experiment is to separate colored pigments from leaves and analyze the components with thin-
layer chromatography.

Learning Objectives
• Explain how Thin Layer Chromatography (TLC) is used in the chemistry lab to separate and identify the
components of chemical mixtures.

• Prepare compounds for TLC analysis.

• Calculate the R𝑓 values of known and unknown compounds and compare against a known standard; and

• Use concepts of intermolecular forces to explain why some compounds have a higher R𝑓 value than others.

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 How Can We Use Chromatography to Separate Plant Pigments?

Procedure

For this experiment, you will work in pairs (groups of 2).

Safety Precautions
• Wear your goggles at all times in the laboratory.
• Wear gloves throughout the experiment.

Equipment and Reagents

Your Equipment Drawer Table Drawers and Bench Top

Two Metal spatulas Forceps

Two Large glass stirring rods Ruler
10 mL Graduated cylinder

Common Equipment Areas Reagent Hood

Two Wide mouth jars 0.5 spatulafuls of Magnesium Sulfate per sample
Three Vials 0.5 spatulafuls of Sand per sample
Three Capillary tubes 40 drops of Acetone per sample
Two TLC silica gel plates Hexane
0.25 g of Spinach Acetone

Bring your own

• One Leaf sample per person (~2 square inches, ~0.5 g; fresh, green, not dried)
• Soft-tip pencil (e.g. #2, non-mechanical)

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 How Can We Use Chromatography to Separate Plant Pigments?

Prepare the Leaf Extracts

1. In the report sheet, briefly describe your leaf sample. Record color, texture, shape, type of plant, etc. Also,
record any observations while preparing the spinach or leaf sample.

2. Tare a paper weigh cup on a balance. Weigh 0.25 g of spinach and place it in
one of the vials provided. Put 1/2 spatula of magnesium sulfate (MgSO4 ) and 1/2
spatula of sand into a weigh cup and transfer the solids to the vial of spinach
(see Figure PP.8).

Figure PP.8: Leaf, MgSO4

3. Grind the mixture in the glass vial with a large stir rod for 2 minutes. and sand sample sizes.

4. Add 40 drops of acetone to the vial and grind for 2 more minutes. Cap the vial and allow the mixture to sit for
10 minutes.

5. Repeat the same preparation with each of your leaf samples. Choose the thinnest,
least veined part of the leaves.

6. You will have three vials: one vial containing spinach and two vials containing your
group’s leaf samples.

Prepare the TLC Plate

You will be using silica gel plates for the separation of the plant pigments. The plate is a Figure PP.9: Vial of
sheet of plastic covered in a thin layer of silica gel. Dry silica gel is a fine white powder leaf extract.
of silicon dioxide. You will be spotting your sample on the silica gel side of the plate. The
silica gel layer is fragile, so try your best to not touch or break the silica gel.

1. Obtain two TLC plates. The TLC plates are in a desiccator on the reagent bench. To open the desiccator, use
one hand to hold the bottom of the desiccator and the other to grasp the top and SLIDE the lid off just enough
to grab a plate. Slide the lid back on the desiccator. Your instructor will demonstrate this technique in the lab.

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 How Can We Use Chromatography to Separate Plant Pigments?

2. Put the TLC plate into the TLC chamber (wide mouth jar) and try to put the cap on. If the plate is too long for
your TLC chamber, it must be cut to size. Use scissors to remove about 5 mm from the short end of the plate. If
the scissors break the silica gel off the plate, grab a new plate.

3. Measure 1 cm from the bottom of the plate and lightly draw a baseline with a pencil (Figure PP.10).

4. Lightly with a pencil make three hash marks approximately 0.7 cm apart along the baseline. (see Figure PP.10).

Figure PP.10: Draw a baseline. Make 3 hash marks along the baseline.

Prepare the Solvent Chambers

1. You will prepare 2 different solvent chambers with 2 different ratios of solvent. You will use a mixture of 4 mL
of acetone and 6 mL of hexane for your first solvent chamber and 10 mL of acetone or 10 mL of hexane for your
second chamber, which will be assigned by your TA.

2. Using a 10-mL graduated cylinders measure the appropriate mixture of solvent and add to a wide mouth jar.

3. Cap the jar with one of the lids provided and swirl the jar to mix the solvents.

4. Set the capped chamber beneath the snorkel hood to saturate with solvent vapor.

5. Repeat with your second wide mouth jar and ratio of solvent.

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Load the TLC Plate (beneath snorkel hood)

1. You will load the samples alternatively at each hash mark or lane so that you have 1 spot of the spinach standard
and 1 spot each of your leaf samples (sample 1 and sample 2). The best practice is to spot (from left to right)
sample 1, spinach, sample 2 across the baseline, so the spinach reference is between the two samples. Label the
identity of the samples in Report Table PP.1.

2. Obtain 3 capillary spotters from your instructor. The spotters are dispensed from the tube through a hole in the
cap. Just turn the tube upside down and shake. Your instructor will demonstrate.

3. Dip one end of spotter into the acetone extract from a sample. The capillary
action of the tube will cause the liquid to fill the tube. Do not pull solid into the
tube. (See Figure PP.11)

4. Briefly and gently press the end of the tube onto the baseline. A small dot will
Figure PP.11: Filling the
form on the plate. It does not need to be large. capillary tube.

5. Allow the spot to dry for 10 seconds.

6. Re-spot the same sample 2 more times in the same place, allowing the spot to dry each time.

7. Spot all samples using the same method. Use a new spotter for each sample. Do not contaminate your sample.

8. Repeat with your second TLC plate.

9. Discard spotters in glass trash.

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 How Can We Use Chromatography to Separate Plant Pigments?

Develop the TLC Plate (beneath snorkel hood)

1. Using forceps, carefully place the TLC plate in the solvent chamber and cap the jar (Fig-
ure PP.12). Take care not to splash the plate with solvent as you are putting it in the
chamber. Make sure the plate is level. Avoid moving the chamber as this can dis-
turb the flow of the mobile phase which in turn can distort your results.

If the level of the solvent is above the baseline, you will have to remake
your TLC plate.

2. While your first TLC plate is developing, repeat step 1 with your second plate.

3. Inspect your plates inside the chamber when the solvent reaches about halfway up the Figure PP.12: Use
plate (Figure PP.13). Can you identify the pigments of the spinach standard? forceps to place the
plate in the solvent
chamber.

Figure PP.13: Half-

developed TLC plate.

4. Allow the solvent to move up the plate until it reaches approximately 1 inch from the
top of the plate.

Do not allow the solvent to reach the end of the plate or else you will have to restart your
TLC.

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▶ The next 2 steps must be done quickly because the pigments can rapidly fade. Have one partner
keep an eye on the second TLC plate to ensure the solvent does not run off the end of the plate.

5. Quickly remove the plate from the chamber using forceps (Figure PP.14).

6. Very quickly trace the solvent front with a pencil. (It is the part of the
plate near the top where the plate will appear “wet”.) Trace the outline
of all of the visible spots with a pencil (Figure PP.15) AND quickly take
a picture of the plate before the pigments fade.

Figure PP.14: Quickly remove

TLC plate with forceps.

Figure PP.15: Circle the visual spots.

7. For each spot, measure and record the distance from the baseline to the
center of the spot and the baseline to the solvent front. The distance that
the pigment traveled divided by the distance traveled by the solvent from
the baseline is known as the retention factor, or R𝑓 value.

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 How Can We Use Chromatography to Separate Plant Pigments?

8. Show your TA your chromatogram. They will tell you if you need to repeat
the separation.

9. You will need to upload a picture of both of your annotated TLC plates and a picture of an annotated TLC plate
from a solvent ratio that you did not test (i.e. if you tested 10 mL of acetone, you will need to get a picture from
another group of the TLC plate that tested 10 mL of hexane).

Waste Disposal and Clean Up

• Use a 250-mL beaker under the snorkel exhaust at your lab table to collect the solvent and sample waste
from the vials and wide-mouth jar.
• Discard the spinach and leaf extract from the vials into this waste collection beaker.
• Use your forceps to remove large pieces of leaf material and dispose in the regular trash.
• Rinse the vials twice with a few mL of water and discard the rinses into the waste beaker as well.
• Discard the vials in the glass disposal box and discard the vial caps in the regular trash.
• Discard the mobile phase from the jar into the waste beaker. Place the wide-mouth jar and lid under your
snorkel hood for a few minutes to allow any excess solvent to evaporate.
• After collecting all the solvent and sample waste in the waste beaker, the contents of the waste beaker can
poured into the Solvent and Sample Waste container in the main hood.
• Discard used spotting tubes in the glass trash box.
• Discard the used TLC plates in the regular trash.
• Return the wide-mouth jars and lids to the cabinet.
• Return the forceps and rulers to the table drawers.

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 Name:
Report Sheet:
Section: Date: How Can We Use Chromatography to Separate Plant Pigments?

Prepare the Leaf Extracts

Provide observations for your samples.
Leaf Sample 1

Leaf Sample 2

Spinach

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 How Can We Use Chromatography to Separate Plant Pigments? Report Sheet

Load the TLC Plate (beneath snorkel hood)

Report Table PP.1: Identity of the Samples in Each Lane of the TLC plate

Lane Sample Identity

Upload photos of three TLC plate pictures.

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 How Can We Use Chromatography to Separate Plant Pigments? Report Sheet

Data Analysis and Results

Upload photos of three TLC plate pictures from the different solvent ratios.
Indicate the sample identities, the origin, the solvent front, and the spots that you circled.
Calculate the R𝑓 value for each component of the spinach standard sample by measuring and recording the distance
(in mm) from the center of the spot to the baseline and the distance (in mm) from the baseline to the solvent front.
Identify each pigment based on comparison to the R𝑓 values provided in the introduction.
Calculate the R𝑓 value for each component in the Leaf samples. Assign a possible identity of each pigment based on
comparison to the spinach standard for both of the Leaf samples. Your leaf samples may contain pigments or spots
that do not have comparable spots in the spinach standard. Label these components as “unknown pigments”.

TLC Data in 4:6 Acetone:Hexane Solvent

Report Table PP.2: TLC Data for Spinach Reference Standard with 4:6 Acetone:Hexane Solvent

Compo- Color Distance Spot Distance R𝑓 Identity

nent traveled Solvent
(mm) traveled
(mm)

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 How Can We Use Chromatography to Separate Plant Pigments? Report Sheet

Report Table PP.3: TLC Data for Leaf Sample 1 with 4:6 Acetone:Hexane Solvent

Compo- Color Distance Spot Distance R𝑓 Identity

nent traveled Solvent
(mm) traveled
(mm)

Report Table PP.4: TLC Data for Leaf Sample 2 with 4:6 Acetone:Hexane Solvent

Compo- Color Distance Spot Distance R𝑓 Identity

nent traveled Solvent
(mm) traveled
(mm)

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 How Can We Use Chromatography to Separate Plant Pigments? Report Sheet

TLC Data in Acetone Solvent

Report Table PP.5: TLC Data for Spinach Reference Standard with Acetone Solvent

Compo- Color Distance Spot Distance R𝑓 Identity

nent traveled Solvent
(mm) traveled
(mm)

Report Table PP.6: TLC Data for Leaf Sample 1 with Acetone Solvent

Compo- Color Distance Spot Distance R𝑓 Identity

nent traveled Solvent
(mm) traveled
(mm)

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 How Can We Use Chromatography to Separate Plant Pigments? Report Sheet

Report Table PP.7: TLC Data for Leaf Sample 2 with Acetone Solvent

Compo- Color Distance Spot Distance R𝑓 Identity

nent traveled Solvent
(mm) traveled
(mm)

TLC Data in Hexane Solvent

Report Table PP.8: TLC Data for Spinach Reference Standard with Hexane Solvent

Compo- Color Distance Spot Distance R𝑓 Identity

nent traveled Solvent
(mm) traveled
(mm)

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 How Can We Use Chromatography to Separate Plant Pigments? Report Sheet

Report Table PP.9: TLC Data for Leaf Sample 1 with Hexane Solvent

Compo- Color Distance Spot Distance R𝑓 Identity

nent traveled Solvent
(mm) traveled
(mm)

Report Table PP.10: TLC Data for Leaf Sample 2 with Hexane Solvent

Compo- Color Distance Spot Distance R𝑓 Identity

nent traveled Solvent
(mm) traveled
(mm)

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Post-Lab Questions

1. List the following pigments in order of most polar to least polar (non-polar): chlorophyll a, chlorophyll b,
xanthophyll, beta-carotene. Justify or explain the basis of your answer.

2. Explain how you identified the components of your samples. Give at least one specific example.

3. Why do you need to run a TLC of the spinach instead of just using the R𝑓 values provided in the lab manual?

4. What differences were there between the spots on your two TLC plates? What caused these differences? Explain
using what you know about TLC and the mobile phases in each experiment.

5. Suppose you conduct a third trial using 8 mL of acetone and 2 mL of hexane as your mobile phase. What
differences would you expect to see in this third TLC plate? Explain using what you know about TLC and the
composition of the mobile phase.

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