Photosynthesis: Question 7

Worked solution

Part (a): Calculating the Rf value of chlorophyll b

The $R_f$ (retardation factor) value of a pigment is defined as:

$R_f = \frac{\text{distance travelled by the pigment}}{\text{distance travelled by the solvent front}}$

For chlorophyll b, the pigment travelled 4.3 cm from the origin, while the solvent front travelled 9.6 cm:

$R_f = \frac{4.3}{9.6} = 0.4479\ldots \approx 0.45$

So the $R_f$ value of chlorophyll b is 0.45 (to 2 decimal places).

Part (b): Why Rf values differ between pigments

Whether a pigment ends up travelling far or only a short distance up the chromatography paper depends on a balance between two things: how soluble it is in the moving, non-polar solvent, and how strongly it is retained (adsorbed) by the stationary paper. A pigment that dissolves more readily in the non-polar solvent spends more of its time moving with the solvent front and so is carried further, giving it a higher $R_f$ value.

• Carotene is a pure hydrocarbon, containing only carbon and hydrogen, so it is strongly non-polar. It therefore dissolves very readily in the non-polar solvent and is carried almost as far as the solvent front itself, giving it the highest $R_f$ of the four pigments.
• Chlorophyll b has additional oxygen-containing functional groups within its structure, making it somewhat more polar than carotene. This makes it less soluble in the non-polar solvent and causes it to be retained more strongly on the paper, so it travels the shortest distance and has the lowest $R_f$.

Part (c): Confirming pigment identity with a second solvent

An $R_f$ value is only meaningful for one specific combination of solvent and stationary phase (paper). It is possible for two chemically different pigments to happen to have very similar solubility in one particular solvent, and so travel to almost the same position, purely by coincidence.

By repeating the chromatography with a second solvent of different polarity, the relative solubility of each pigment is likely to change, so pigments that separated poorly (or overlapped) in the first solvent may separate more clearly in the second, and vice versa. If the unknown spot lines up with a known chlorophyll a reference spot’s position in the second solvent as well as in the first, this is strong evidence that the two spots are the same pigment. If instead the unknown spot moves to a different position from the chlorophyll a reference in the second solvent, this shows they are in fact different pigments that simply had a coincidentally similar $R_f$ in the first solvent.

Final answers

• (a) $R_f$ of chlorophyll b $= 4.3 / 9.6 = 0.45$.
• (b) Carotene is strongly non-polar so is highly soluble in the non-polar solvent and travels furthest (highest $R_f$); chlorophyll b is more polar, is less soluble and is retained more by the paper, so it travels least (lowest $R_f$).
• (c) Rf values are specific to a solvent/paper combination, so running a second, different solvent and checking whether the unknown spot still matches the chlorophyll a reference confirms (or rules out) its identity.