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Unit 2 Aim C
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Unit 2- Learning aim CUndertaking chromatographic techniques to identify components in mixtures
During this assignment, I will be carrying out various chromatographic techniques in order to separate
and identify components in mixtures. This will include paper chromatography of chlorophyll and amino
acids as well as thin-layer chromatography of chlorophyll. I will then analyse my results from these
chromatographic techniques and will evaluate the links between the chromatography techniques used
and the chromatogram produced suggesting methods of improvement where required in order to
obtain better results.
Introduction-
Chromatography is the separation of components in a mixture. To begin the process, the mixture is
dissolved in a substance known as the mobile phase, which is the solvent in paper chromatography and
thin-layer chromatography, which transports it through a second substance known as the stationary
phase which is the paper in paper chromatography or the TLC plate in thin-layer chromatography. The
different components of the mixture travel at different speeds through the stationary phase, forcing
them to separate from one another. The specific mobile and stationary phases determine which
chemicals travel faster or slower and how they are separated. The distance that the solvent travels up
the paper indicates how soluble the substance is. The further it travels up the stationary phase, the
more soluble it is and as a result, the less attractive.
A chromatogram is the result of chromatographic separation. A paper chromatogram can be used to
differentiate between pure and impure compounds; a pure material will create one spot on the
chromatogram, whereas an impure substance, or mixture, will produce two or more spots. A paper
chromatogram can also be used to detect compounds by comparing them to already identified
substances. If two substances create the same number of dots that match in colour or go the same
distance up the paper, they are likely to be the same.
The size and angle of the spots on the chromatography paper can provide valuable information about
the compounds present in the mixture. There are several factors that can affect the size and angle of the
spots in paper chromatography, including:
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,Solubility: The solubility of a compound in the solvent used in the chromatography can affect the size
and shape of the spot. Compounds that are more soluble will tend to spread out more, resulting in
larger spots. Less soluble compounds will tend to remain in a smaller area, resulting in smaller spots.
Adsorption: The chromatography paper used in the procedure can also impact the size and form of the
spots. Certain papers have a higher adsorption capacity than others, resulting in smaller spots as a result
of increased adsorption.
Flow rate: The rate at which the solvent moves through the paper can influence the size and shape of
the spots. Faster flow rates can produce larger spots, whereas slower flow rates can produce smaller
spots.
Concentration: The concentration of the compounds in the mixture can also affect the size and shape of
the spots. Compounds that are more concentrated will tend to produce larger spots, while less
concentrated compounds will produce smaller spots.
Shape of the container: The shape of the container used to develop the chromatogram can affect the
angle of the spots. A container with a circular base will produce circular spots, while a container with a
rectangular base will produce rectangular spots.
Rf value-
The retention factor (Rf) is used in chromatography to compare and identify substances. The Rf value of
a compound is equal to the compound's distance travelled divided by the solvent front's distance
travelled both measured from the origin.
Page 2 of 22
, Types of chromatography-
There are various types of chromatography however, some of the main types include gas
chromatography, liquid chromatography, thin- layer chromatography and ion exchange
chromatography.
In gas chromatography, the components of a sample are dissolved in a solvent, evaporated, and passed
through a stationary phase with an inert gas, generally nitrogen or helium. Larger molecules take longer
to move through the column and reach the detector at the other end.
Liquid chromatography uses a solid stationary phase to separate molecules in a liquid mobile phase. The
mixture of interest is dissolved in a liquid and passed through a solid stationary phase, which is generally
made of silica.
The stationary phase in thin-layer chromatography is a thin layer of material, commonly silica-based,
and the mobile phase is a liquid in which the mixture is dissolved in. As the combination goes through
the stationary phase, the various components will separate at different stages. Because of the polarity of
the chemicals used, the components have varying degrees of attraction to both the mobile and
stationary phases.
Ion exchange chromatography separates the components of a mixture based on their charge, in addition
to or instead of their size. In essence, positively (cations) or negatively (anions) charged ions are
separated using different stationary phases and different pH mobile phases. (1)
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