beaker
M
X
Y
Z
base line
spots of dye on chromatography paper
solvent
M
X
Y
Z
the finished chromatogram
chromatography apparatus
Unit 3 Methods of purification
solvent front
Lid
Figure 3.01 • Draw a pencil line on the chromatography paper.
• Put spots X, Y and Z that you think are in the dye mixture on the base line for comparison. • Dip the bottom of the paper in the solvent. • The solvent moves up the paper and allows separation of the mixture. • Remove the paper when the solvent front is near the top. The spots on the chromatogram can be compared with those of known dyes. In Figure 3.01, the mixture, M, contains dyes X and Z as well as a third dye.
TIP
We can identify the components in the mixture because for a particular solvent they travel a certain distance compared with the solvent front. When carrying out chromatography, the solvent level should be below the base line to prevent the mixture washing off into the solvent.
Rf =
distance from base line to centre of spot distance of solvent front from base line
Rf values can be used to identify compounds or ions because most substances have characteristic Rf values.
Locating agents TERMS Locating agent: A substance that reacts with colourless spots on a chromatogram to make them visible as coloured spots. Many compounds, for example amino acids, are colourless and so cannot be seen on a chromatogram. Spraying the chromatogram with ninhydrin and warming makes the spots appear a purple colour. Ninhydrin is an example of a locating agent.
TIP
• Put a spot of the concentrated mixture to be separated, M, on the line using a very thin tube.
You do not have to know the names of particular locating agents.
Retention factor (Rf) TERMS Rf : In chromatography, the distance moved by a particular substance from the base line divided by the distance moved by the solvent front from the base line.
Worked example 3.01 Plant leaves contain pigments called chlorophylls. Describe how you would make a solution of these pigments and use chromatography to separate them. [8]
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