Chromatography Practical 1

Practical 1: Chromatography
Preparation and evaluation
Prepare for a short test based on Experiment 1. Access videos from the electronic version of this
experiment on the NCHE 121 eFundi website.

Experiment 1: Analysis of pigments with thin layer chromatography

1.1.1. Background
Plants are valuable sources of chemical compounds that are generally known as natural products.
To obtain these compounds in the pure form usually involve many complex steps that may take a long
time to complete. In this experiment you will isolate and identify pigments from spinach.

Vegetables make four main types of pigments to colour themselves. The most stable pigments are the
carotenoids. These compounds are found in yellow or orange fruits and vegetables, such as tomato,
carrots, maize, pumpkin, sweet potato, peaches, and citrus fruits. There are also a few examples of
reddish carotenoids in apricots, persimmons, pink grapefruit, paprika, red pepper, and tomato. The
carotenoids are divided into two main groups – the carotenes and the xanthophylls.

Figure 1-1: The structure of β-carotene – an example of a carotene

Xanthophylls are structurally similar to carotenes but have additional hydroxyl groups.

Figure 1-2: The structure of a xanthophyll

Carotenoids are tough – they are resistant to heat and acidity and can be overcooked without much
loss of colour. Although the carotenoids are present in all green vegetables and in green leaves on
trees, their colours are masked by the dominant green chlorophylls. The green colour of the
chlorophylls fades in autumn and then the more stable carotenes show off their pretty yellow, orange
and red colours.

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 N
R
N Mg
O N

N
O O

O
O

Figure 1-3: The structures of chlorophyll a (R = CH3) and chlorophyll b (R = CHO)

The green vegetables are the most prevalent in our diet and include asparagus, broccoli, cabbage,
celery, eggplant, green beans, green peppers, peas, and spinach. Pheophytins are greyish pigments
that are formed when the central Mg2+ ion is removed from the chlorophylls. These compounds form
easily when chlorophylls are placed in acidic solutions, such as vinegar. The red and blue colours of
beets, red cabbage, red onions, and most berries are caused by anthocyanins. A fourth pigment
group known as anthoxanthins produces the creamy whites of cauliflower, onions, parsnips, rice, and
white cabbage. Spinach leaves are a convenient source of pigments. It contains carotenoids,
chlorophylls, and pheophytins. In this experiment, you will attempt to separate these compounds by
chromatography. The different colours of the compounds will enable you to visually follow the progress
of the separation.

1.1.2. New experimental skills required in this experiment

1.1.2.1. Determining mass with an analytical balance

An analytical balance is essential in a chemistry laboratory. Our laboratory is equipped with balances
that can display three decimal places. Readings should, therefore, be taken and reported to the
nearest 0.001 g. The video shows a balance that can measure to four decimal places. A given type of
balance has a limit on the mass that it can weigh. The larger the maximum mass that a balance can
weigh, the fewer the number of decimal places it can display. Make sure that you can TARE a balance
and understand what this means.

Using an analytical balance

1.1.2.2. Solid-liquid extraction

Using a solvent to selectively dissolve some components of a mixture of solids is known as solid-
liquid extraction. With this method, one or more soluble solids can be separated from insoluble
materials. Making a cup of tea involves extraction of caffeine and other organic compounds from the
tea leaves with hot water. The hot water selectively dissolves these compounds leaving the tea leaves
behind. In this experiment, you will use acetone to dissolve some pigments from spinach leaves. The
procedure is an example of solid-liquid extraction.

1.1.2.3. Chromatography

To separate the different pigments in a spinach extract, you will need to use a separation method
known as chromatography.

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During chromatography, a mixture of compounds flows over or through a material which slows
down the compounds differently by some interaction. The components of the mixture therefore
do not move at the same speed and are therefore separated from each other.

The chromatography that you will use in this experiment is known as liquid chromatography (LC)
because the mixture is dissolved in a liquid. There are different types of liquid chromatography based
on the type of interaction between the liquid and the material through which it flows. In this experiment,
we will only focus on chromatography based on adsorption. Figure 1-4 explains the difference
between absorption and adsorption. A compound is absorbed when it enters a surface, for example
when water enters the pores in a sponge. A compound is adsorbed when a compound sticks to a
surface due to intermolecular forces.

Figure 1-4: The difference between absorption and adsorption

During adsorption chromatography, a solvent containing a mixture of components (compounds,

pigments) moves over a solid material. The moving solution is known as the mobile phase and the
solid material is called the stationary phase. Components of the mixture will have different abilities to
adsorb (stick to) the stationary phase and to dissolve in the mobile phase. These different affinities for
the mobile and stationary phases are caused by intermolecular forces of different magnitudes existing
between the components and the different phases. A component with a higher affinity for the mobile
phase is dissolved in the solvent for longer than it is adsorbed onto the stationary phase. Such a
component moves over the stationary phase quicker. A compound that adsorbs better to the
stationary phase than it dissolves in the mobile phase will move along slower. The different speeds at
which the components move over the stationary phase account for the separation by chromatography.

Separation by adsorption can be achieved by placing the stationary phase into a column. The
separation process is illustrated in Figure 1-5. This method is sometimes called column
chromatography. The mobile phase is poured into the column and it moves through the stationary
phase because of gravity. Look at the following videos to see column chromatography in action.

Column chromatography

Column chromatography of spinach extract

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 Figure 1-5: Separating a mixture with a chromatographic column

In this practical session, you will use a different form of adsorption chromatography known as thin
layer chromatography (TLC). In this type of chromatography, the stationary phase is a solid that was
coated onto a glass or plastic plate. The mobile phase moves through the stationary phase due to
capillary action. Imagine that you take some of the silica stationary phase used in the chromatographic
column and apply it onto a glass or plastic plate so that it forms a thin layer. Look at the following
videos to see TLC in action.

The basics of doing as TLC analysis

TLC of spinach fractions

The separation of components can be improved by using solvents with different polarities. For
example, a polar compound will have a higher affinity for a more polar mobile phase and will move
over the stationary phase faster. A mixture of solvents with different polarities can also be used to
achieve a better separation. For example, in today's experiment a mixture of 1 part acetone (more
polar) and 4 parts hexane (non-polar) is used as the mobile phase.

Using a polar solvent will increase the rate at which all components move over a polar stationary
phase. This is due to the solvent hydrogen bonding to the surface of the stationary phase. This makes
the stationary phase less polar because the bonded solvent blocks some of the polar groups on its
surface. Using a polar solvent, such as acetone, provides a way to "wash" very polar compounds out
of the column. The effect of a polar solvent is illustrated in the following video.

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 Influence of mobile phase polarity in this experiment

A good simulation of the influence of solvent polarity on a TLC analysis is available here.

Different stationary phases are available. The most popular choices for adsorption chromatography
are silica gel (silicon dioxide) and alumina (aluminium oxide). Both these stationary phases have polar
groups and have a higher affinity for the polar components of a mixture. If we use a polar stationary
phase and non-polar mobile phase, we can expect that the components of a mixture will reach the end
of the column in order of increasing polarity. {The most polar component will move through the column
the slowest because it has the highest affinity for the polar stationary phase.}

When a relatively non-polar mobile phase is used, the non-polar components of a mixture will
move through the polar column faster than the polar components.

It will not always be possible to isolate pure pigments when using column chromatography, especially
when the structures of the components are very similar. In such cases, collected fractions may contain
more than one pigment compound. Thin layer chromatography can be used to examine the different
fractions collected from a column chromatography experiment. The TLC analysis will indicate if a given
fraction contains a single component or more than one component. More sophisticated instrumental
chromatographic methods can also be used to reach the same goal.

1.1.3. Experimental method

Additional apparatus and consumables

• Propette
• UV light
• Thin layer chromatography plates
• Glass capillary tubes
Chemicals
• Spinach leaves
• Acetone
• 1:1 mixture of petroleum ether and acetone

1.1.3.1. Preparing the spinach extract

1. Weigh out approximately 5 g of spinach leaf pieces. Tear out the soft parts – do not use the stems
and veins of the leave.

2. Take the spinach pieces to your workstation. Tear them into smaller pieces [about 5 mm2] and
place them into a 250 cm3 beaker.

3. Add 10 cm3 of acetone to the beaker and mix the contents with a spatula. Make sure that the
acetone contacts all the leaf pieces. Stir the mixture for 2 minutes. {This is a washing procedure
that removes the protective wax layer on the spinach leaves.}

Acetone is flammable.

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4. Pour the acetone from the spinach leaves. Do this in such a way that the spinach leaves stay
behind. (This procedure is known as decantation.)

5. Add 8 cm3 of acetone to the beaker. Crush, grind and stir the spinach leaf pieces with a spatula for
at least 10 minutes. The acetone will turn dark green during this time.

6. Allow the mixture to stand for 10 minutes. Stir and grind the spinach leaf pieces occasionally.

7. Decant the acetone solution from the spinach leaves and into a clean, dry porcelain dish. You may
use your spatula to keep the spinach leaves in the beaker and to press additional acetone solution
from the leave pieces.

Your spinach extract contains water which may influence the outcome of the TLC analysis. To
compare the results of TLC analysis with wet and dry spinach extract, you will receive dried
spinach extract from your student assistant.

1.1.3.2. Thin layer chromatography [TLC]

1. Get a TLC plate from your student assistant.

2. Draw a light pencil line about 1 cm from the bottom of the plate [Figure 1-6].

Figure 1-6: Preparing the TLC plate for use

3. Use a small beaker to collect ~1 cm3 of dried spinach extract from your student assistant.

4. Use a capillary tube to "spot" some of the wet spinach extract in the porcelain dish onto the TLC
plate in the position labelled A [Figure 1-7]. The spot should be small – approximately 3 mm in
diameter. You may make the spot darker by allowing the original spot to dry and then spotting the
same mixture at the same place on the TLC plate.

5. In the same way, spot the dry spinach extract in position B on the TLC plate. Use a new capillary
tube to apply the spot of each extract.

To prevent contamination, a new capillary tube must be used to apply the spot of each sample.

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 Figure 1-7: Spotting the spinach extract on the TLC plate

6. Prepare a development chamber for the TLC plate by placing a filter paper against the inside wall
of a 250 cm3 beaker. Figure 1-8 shows the layout of the development chamber without the filter
paper. Add 10 cm3 acetone-petroleum ether mixture to the beaker and cover it with a watch glass.

Figure 1-8: Development chamber for a TLC plate

Acetone and petroleum ether are flammable.

7. Carefully lower the TLC plate into the development chamber. The solvent will move up the plate
and separate the different components of the spots. Let the TLC plate develop until the solvent
has moved to about 0.5 cm from the top of the plate. Remove the plate and immediately draw
a horizontal pencil line to mark the position of the solvent front with a pencil. {The solvent front is
the level to which the solvent has moved up the TLC plate.}

8. Allow the TLC plate to dry and examine it under visible light and encircle any spots. Then examine
your plate under ultraviolet (UV) light and mark any additional spots. Indicate which spots are only
visible under UV light if any.

9. Use the developed TLC plate to determine the number of components in each of the fractions that
you obtained from the column chromatography procedure.

10. You can use Figure 1-9 to help you interpret the TLC analysis.

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 Figure 1-9: A key to the TLC analysis

1.1.4. Waste disposal and clean-up

• Chemical substances that were used or produced in this experiment should be discarded into the
indicated waste containers.

• TLC plates can be discarded in the solid waste bin.

• Used capillary tubes must be discarded in the broken glass container.

• Wash and pack away all your apparatus. Ensure that your workspace is clean and dry and that all
water and gas taps are closed.

1.1.5. Experimental report

Complete the report and submit it to your student assistant before you leave the laboratory.

Perspective on your results

Obtaining pure substances is essential in chemistry because it enable the chemist to determine the
identities of compound. This is because every pure substance has a unique set of physical and
chemical properties. The compositions of plant materials are extremely complex. Separating a mixture
of many compounds may be a challenge. One strategy to obtain pure substances is to subject the
fractions of the first chromatographic separation to a second chromatographic separation. Further
chromatographic separations can be performed until pure substances are obtained.

Link to later chemistry

Chromatography is the analytical method used most in chemistry. Machines equipped with thin and
usually long columns are used to separate complex mixtures. These machines can be calibrated so
that the substances in the mixture can be quantified or linked to other machines that determines the
identities of the different substances.

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Figure 1-10 shows a high-performance liquid chromatograph (HPLC).

Figure 1-10: A modern HPLC machine (or instrument) used in liquid chromatography

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