Using Chromatography to Separate Substances for Identification
Chromatography is a process used to separate components in a mixture, using a stationary phase
and a mobile phase. There are many different types of chromatography, the most common being
paper chromatography, thin layer chromatography and column chromatography; however, they all
use a stationary phase and a mobile phase. (1) This technique has a variety of uses, for example:
drug testing and manufacture, food industry, assessing pollution, forensic pathology and insulin
purification. (2)
Paper Chromatography of Plant Pigments
Paper chromatography aims to separate mixtures of soluble substances, such as inks, dyes and plant
pigments. Solvents such as water or propanone are used as the mobile phase to carry the mixture
through the stationary phase – chromatography paper. (3) This paper is made from fibres of
cellulose – a polymer of glucose. During manufacture, water vapour in the atmosphere binds to the
surface of the paper, due to the OH group seen below. This layer of water is what allows the paper
to work in chromatography. Solvents used can be polar or non-polar. When using a non-polar
solvent (polarity index below 15), for example hexane, there will be little attraction between the
non-polar molecules in the solvent and the water molecules in the paper, so will spend most of its
time moving up the paper, being carried by the solvent as it travels, it will therefore have a relatively
high Rf value. Using a polar solvent such as water, there will be strong attraction between the polar
molecules in the solvent and the water molecules in the paper – this is because water molecules are
also polar, and like attracts like. Due to this attraction, the polar molecules will mostly dissolve in the
water on the surface of the paper, spending less time in the mobile phase, and therefore moving less
distance. This gives them a lower Rf value. (4) The degree of polarity of the different molecules in a
mixture will determine how far the molecules travel up the paper, separating them from one
another. The Rf (retention factor) is an arbitrary measurement of the distance travelled by molecules
up the paper, it is calculated using the equation Rf = distance travelled by pigment / distance
travelled by solvent front.
Figure 1. Cellulose structure, (6)
, Method: See lab book.
Table 1: Separation of Plant Pigments using Paper Chromatography
Solvent Distance Travelled by Distance Travelled by Rf Value
Solvent (cm) Pigment (cm)
Distilled water 10.0 0.0 0.0
Petroleum ether 9.5 1.3 0.1
Propanone 9.5 9.0 0.9
Petroleum Ether : 11.4 10.4 0.9
propanone 7 : 3
Figure 2. Results of Paper Chromatography of Plant Pigments
The mobile phase which produced the best results was petroleum ether, it separated the pigments
out the most clearly, and had an Rf value of 0.1 as seen in table 1 above. The pigments in the spinach
leaves used are chlorophyll a and chlorophyll b, these pigments were identified within the
chromatograms using their colour, relative positions and Rf value ranges. The two chlorophyll
pigments, a and b, are identical in structure, besides the functional group of their third carbons, as
seen in Figure 3 below. This general structure makes both chlorophyll a and b non-polar pigments
(8); however, the methyl group of chlorophyll a contains three hydrogen atoms bound to a single
Chromatography is a process used to separate components in a mixture, using a stationary phase
and a mobile phase. There are many different types of chromatography, the most common being
paper chromatography, thin layer chromatography and column chromatography; however, they all
use a stationary phase and a mobile phase. (1) This technique has a variety of uses, for example:
drug testing and manufacture, food industry, assessing pollution, forensic pathology and insulin
purification. (2)
Paper Chromatography of Plant Pigments
Paper chromatography aims to separate mixtures of soluble substances, such as inks, dyes and plant
pigments. Solvents such as water or propanone are used as the mobile phase to carry the mixture
through the stationary phase – chromatography paper. (3) This paper is made from fibres of
cellulose – a polymer of glucose. During manufacture, water vapour in the atmosphere binds to the
surface of the paper, due to the OH group seen below. This layer of water is what allows the paper
to work in chromatography. Solvents used can be polar or non-polar. When using a non-polar
solvent (polarity index below 15), for example hexane, there will be little attraction between the
non-polar molecules in the solvent and the water molecules in the paper, so will spend most of its
time moving up the paper, being carried by the solvent as it travels, it will therefore have a relatively
high Rf value. Using a polar solvent such as water, there will be strong attraction between the polar
molecules in the solvent and the water molecules in the paper – this is because water molecules are
also polar, and like attracts like. Due to this attraction, the polar molecules will mostly dissolve in the
water on the surface of the paper, spending less time in the mobile phase, and therefore moving less
distance. This gives them a lower Rf value. (4) The degree of polarity of the different molecules in a
mixture will determine how far the molecules travel up the paper, separating them from one
another. The Rf (retention factor) is an arbitrary measurement of the distance travelled by molecules
up the paper, it is calculated using the equation Rf = distance travelled by pigment / distance
travelled by solvent front.
Figure 1. Cellulose structure, (6)
, Method: See lab book.
Table 1: Separation of Plant Pigments using Paper Chromatography
Solvent Distance Travelled by Distance Travelled by Rf Value
Solvent (cm) Pigment (cm)
Distilled water 10.0 0.0 0.0
Petroleum ether 9.5 1.3 0.1
Propanone 9.5 9.0 0.9
Petroleum Ether : 11.4 10.4 0.9
propanone 7 : 3
Figure 2. Results of Paper Chromatography of Plant Pigments
The mobile phase which produced the best results was petroleum ether, it separated the pigments
out the most clearly, and had an Rf value of 0.1 as seen in table 1 above. The pigments in the spinach
leaves used are chlorophyll a and chlorophyll b, these pigments were identified within the
chromatograms using their colour, relative positions and Rf value ranges. The two chlorophyll
pigments, a and b, are identical in structure, besides the functional group of their third carbons, as
seen in Figure 3 below. This general structure makes both chlorophyll a and b non-polar pigments
(8); however, the methyl group of chlorophyll a contains three hydrogen atoms bound to a single
