Chlorophyll & Other Plant Pigments (AQA GCSE Combined Science: Synergy: Life & Environmental Sciences): Revision Note

Chromatography

Separating pigments in plant leaves using chromatography

• Chromatography can be used to separate and identify chloroplast pigments that have been extracted from a leaf e.g. chlorophyll

• Chromatography works by separating substances with different solubilities using a solvent

• It uses supporting medium - usually paper, to produce a chromatogram that can be studied to identify the substances

  • A pencil line is drawn on chromatography paper and spots of the sample are placed on it

  • Pencil is used for this as ink would run into the chromatogram along with the sample

    • The paper is then lowered into the solvent container, making sure that the pencil line sits above the level of the solvent so the samples don’t wash into the solvent container

  • The solvent travels up the paper by capillary action, taking some of the coloured substances with it

• Different substances have different solubilities so will travel at different rates, causing the substances to spread apart

• Those substances with higher solubility will travel further than the others

The pigments in ink can be analysed using paper chromatography

The mobile and stationary phase

• All chromatography techniques use two phases called the mobile phase and the stationary phase

• In paper chromatography:

  • The mobile phase is the solvent in which the sample molecules can move, which in paper chromatography is liquid e.g. water or ethanol

  • The stationary phase in paper chromatography is the actual chromatography paper itself

• The substances which are more soluble in the solvent will travel further up the paper because they spend more time in the mobile phase and are thus carried further up the paper than the less soluble components

Distinguishing pure & impure substances

• Pure substances will produce only one spot on the chromatogram

• If two or more substances are the same, they will produce identical chromatograms

• If the substance is a mixture, it will separate on the paper to show all the different components as separate spots

• An impure substance therefore will produce a chromatogram with more than one spot

Diagram showing the analysis of a mixture and pure substances using chromatography

Rf value

Calculating R_f values

• These values are used to identify the components of mixtures

• The R_f value of a particular compound is always the same but it is dependent, however, on the solvent used

• If the solvent is changed then the value changes

• Calculating the R_f value allows chemists to identify unknown substances because it can be compared with R_f values of known substances under the same conditions

• These values are known as reference values

• Retention factor, R_f, is calculated by the equation:

• The R_f value is a ratio and therefore has no units and will be less that 1

Calculating Rf value

Using R_f values to identify components of a mixture

• The R_f value of the substances in the chromatogram above can be calculated by: