Unit 2: Practical Scientific Procedures and Techniques
Learning aim C: Undertake chromatographic techniques to identify components in mixtures
Separate to identify
As part of my induction period as a newly appointed technical assistant at a chemical plant, I undertook different
chromatography experiments to separate and identify components in a mixture. The chromatography methods that
were carried out were paper chromatography of spinach leaves, thin layer chromatography (TLC) of spinach leaves
and paper chromatography of amino acids.
Uses of chromatography and how it is carried out
Components in a mixture are separated using chromatography according to their solubility. There are two phases
that it consists of; a mobile phase and also a stationary phase. When a mixture dissolves in a substance, it enters the
mobile phase and during the stationary phase, it moves through a second substance. Ion exchange
chromatography, high-performance liquid chromatography (HPLC) and gas chromatography are further
chromatography methods. Scientists utilise chromatography to separate organic and inorganic molecules. These
applications include drug testing, forensic testing, food testing and within the preparation of pharmaceuticals.
Factors affecting separation of components in chromatography
The polarity of the molecules can influence how far the compounds travel depending on the type of solvent used.
Polar molecules will be more powerful at attracting polar solvents and will move further if a polar solvent is used
instead of a non-polar solvent. This is because it competes better with the stationary phase, leading to a high R f
value. The molecule size can have a direct impact on the rate at which a substance dissolves. Smaller molecules
dissolve faster in solvents because they can fit into the inter-spatial areas of the solvent's larger molecules [1].
Larger molecules will take longer to dissolve in a solvent because they are not as soluble as small molecules.
Temperature also has a significant impact on solid solubility. As temperature increases, solutes in solvents lose their
bonding strength due to an increase in the kinetic energy of the solvent molecules and the solid becomes more
soluble [1].
Paper chromatography
Like with TLC, paper chromatography is used to separate mixtures and identify substances. In paper
chromatography, the stationary phase is the paper which is quite absorbent and the mobile phase is a liquid solvent
or a mixture of solvents which are used to allow the sample to travel up through the paper. The mobile phase flows
through the stationary phase and carries the components of the mixture with it. Different components travel at
different rates due to several factors such as the type of solvent used, the viscosity of the paper used and the
concentration of the sample [2].
Thin layer chromatography (TLC)
Thin layer chromatography is used to analyse fibres in inks, dyes and paints. It is also used to separate pesticides
and insecticides in food products. In thin layer chromatography (TLC), the mobile phase is an organic solvent and
the stationary phase is a thin layer of silica gel supported on a flat, unreactive surface. Silica is an inert substance
and is used as it is very polar. It also often contains a substance which fluoresces in UV light. This means that if you
shine UV light on the plate, it will all glow apart from where the spots are; the spots will show as darker patches [3].
1
, Unit 2: Practical Scientific Procedures and Techniques
Learning aim C: Undertake chromatographic techniques to identify components in mixtures
Evaluating the uses of paper chromatography and thin layer chromatography (TLC)
Advantages Disadvantages
Paper chromatography Paper chromatography is easy Volatile substances and complex
to set up. It can also identify mixtures cannot be separated.
organic and inorganic Paper chromatography is less
compounds in a mixture. [4] accurate than TLC. [4]
Thin layer TLC tends to produce more Some of the TLC plates do not have
chromatography (TLC) useful chromatograms than long stationary phases. It therefore
paper chromatography. The poses a limitation for the length of
mobile phase moves quicker separation of the mixture. The
and more evenly through the coating on the plate surface is not
stationary phase. There are a uniform, which can lead to
range of absorbancies for the differences in results from the
stationary phase. [5] experiment. [6]
What are the similarities and differences between both chromatographic
techniques?
Both techniques are used to separate and identify the components in mixtures. They both include a liquid mobile
phase and a solid stationary phase. The polarity of the molecules in the stationary and mobile phases determines
how the particles are separated in both types. In thin layer chromatography, adsorption type is the main process for
particle separation, whereas partition type is the process used in paper chromatography. Both techniques have
different stationary phases. In thin layer chromatography, a glass plate coated in silica gel acts as the stationary
phase, and in paper chromatography, the stationary phase is the water trapped in the cellulose filter paper. Thin
layer chromatography takes longer to provide results while paper chromatography requires less time for particle
separation [7].
Paper chromatography of spinach leaves
Risk Assessment
Risk Hazard Precautions Size of risk What to do in the event of an
accident
Ethoxyethane Causes burns. Wear eye protection Medium Measure out the ethoxyethane well away
Flammable. from any sources of ignition and store in
capped bottles to avoid the escape of
flammable vapours. [8]
Petroleum ether Skin irritation Wear gloves Low Wash off immediately with plenty of soap
and water. Seek medical attention if
irritation or symptoms persist. [9]
2
Learning aim C: Undertake chromatographic techniques to identify components in mixtures
Separate to identify
As part of my induction period as a newly appointed technical assistant at a chemical plant, I undertook different
chromatography experiments to separate and identify components in a mixture. The chromatography methods that
were carried out were paper chromatography of spinach leaves, thin layer chromatography (TLC) of spinach leaves
and paper chromatography of amino acids.
Uses of chromatography and how it is carried out
Components in a mixture are separated using chromatography according to their solubility. There are two phases
that it consists of; a mobile phase and also a stationary phase. When a mixture dissolves in a substance, it enters the
mobile phase and during the stationary phase, it moves through a second substance. Ion exchange
chromatography, high-performance liquid chromatography (HPLC) and gas chromatography are further
chromatography methods. Scientists utilise chromatography to separate organic and inorganic molecules. These
applications include drug testing, forensic testing, food testing and within the preparation of pharmaceuticals.
Factors affecting separation of components in chromatography
The polarity of the molecules can influence how far the compounds travel depending on the type of solvent used.
Polar molecules will be more powerful at attracting polar solvents and will move further if a polar solvent is used
instead of a non-polar solvent. This is because it competes better with the stationary phase, leading to a high R f
value. The molecule size can have a direct impact on the rate at which a substance dissolves. Smaller molecules
dissolve faster in solvents because they can fit into the inter-spatial areas of the solvent's larger molecules [1].
Larger molecules will take longer to dissolve in a solvent because they are not as soluble as small molecules.
Temperature also has a significant impact on solid solubility. As temperature increases, solutes in solvents lose their
bonding strength due to an increase in the kinetic energy of the solvent molecules and the solid becomes more
soluble [1].
Paper chromatography
Like with TLC, paper chromatography is used to separate mixtures and identify substances. In paper
chromatography, the stationary phase is the paper which is quite absorbent and the mobile phase is a liquid solvent
or a mixture of solvents which are used to allow the sample to travel up through the paper. The mobile phase flows
through the stationary phase and carries the components of the mixture with it. Different components travel at
different rates due to several factors such as the type of solvent used, the viscosity of the paper used and the
concentration of the sample [2].
Thin layer chromatography (TLC)
Thin layer chromatography is used to analyse fibres in inks, dyes and paints. It is also used to separate pesticides
and insecticides in food products. In thin layer chromatography (TLC), the mobile phase is an organic solvent and
the stationary phase is a thin layer of silica gel supported on a flat, unreactive surface. Silica is an inert substance
and is used as it is very polar. It also often contains a substance which fluoresces in UV light. This means that if you
shine UV light on the plate, it will all glow apart from where the spots are; the spots will show as darker patches [3].
1
, Unit 2: Practical Scientific Procedures and Techniques
Learning aim C: Undertake chromatographic techniques to identify components in mixtures
Evaluating the uses of paper chromatography and thin layer chromatography (TLC)
Advantages Disadvantages
Paper chromatography Paper chromatography is easy Volatile substances and complex
to set up. It can also identify mixtures cannot be separated.
organic and inorganic Paper chromatography is less
compounds in a mixture. [4] accurate than TLC. [4]
Thin layer TLC tends to produce more Some of the TLC plates do not have
chromatography (TLC) useful chromatograms than long stationary phases. It therefore
paper chromatography. The poses a limitation for the length of
mobile phase moves quicker separation of the mixture. The
and more evenly through the coating on the plate surface is not
stationary phase. There are a uniform, which can lead to
range of absorbancies for the differences in results from the
stationary phase. [5] experiment. [6]
What are the similarities and differences between both chromatographic
techniques?
Both techniques are used to separate and identify the components in mixtures. They both include a liquid mobile
phase and a solid stationary phase. The polarity of the molecules in the stationary and mobile phases determines
how the particles are separated in both types. In thin layer chromatography, adsorption type is the main process for
particle separation, whereas partition type is the process used in paper chromatography. Both techniques have
different stationary phases. In thin layer chromatography, a glass plate coated in silica gel acts as the stationary
phase, and in paper chromatography, the stationary phase is the water trapped in the cellulose filter paper. Thin
layer chromatography takes longer to provide results while paper chromatography requires less time for particle
separation [7].
Paper chromatography of spinach leaves
Risk Assessment
Risk Hazard Precautions Size of risk What to do in the event of an
accident
Ethoxyethane Causes burns. Wear eye protection Medium Measure out the ethoxyethane well away
Flammable. from any sources of ignition and store in
capped bottles to avoid the escape of
flammable vapours. [8]
Petroleum ether Skin irritation Wear gloves Low Wash off immediately with plenty of soap
and water. Seek medical attention if
irritation or symptoms persist. [9]
2
