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Photosynthetic Pigments

High schoolBiologyIllustrative normal-phase pigment partitioning with square-root solvent advance and direct Rf ruler measurements

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About this experiment

Develop a four-pigment leaf mixture, mark the solvent front and measure migration distances directly on the chromatogram. Compare stationary phases, solvent polarity and loading while separating model values from your own ruler readings.

Learning goals

  • Measure both distances from the original sample line.
  • Calculate Rf and distinguish it from travel distance.
  • Explain why loading and solvent conditions affect separation quality.

How to use it

  1. 1Develop the starting mixture, stop and mark the solvent front.
  2. 2Select a pigment and drag the two ruler handles to measure its Rf.
  3. 3Record the result before resuming development or changing the mixture.
  4. 4Compare a more polar solvent, another stationary phase and heavier sample loading.

The model

Both distances use the same sample origin.
The stated partition coefficient controls the illustrative migration fraction.

Assumptions

  • Four named pigments have illustrative reference Rf values of 0.95, 0.65, 0.50 and 0.35. They are not universal identification standards or measured constants for a stated solvent.
  • A normal-phase partition rule makes higher solvent polarity lower retention. The alternate adsorbent plate multiplies retention coefficients by 1.4.
  • The solvent front advances with square-root time and stops at 80 mm. Every band centre remains behind that front; Gaussian-like displayed bands broaden with time and loading.
  • Ruler distances are editable measurements, distinct from model partition values. Ratios above one prompt a placement check.
  • Changing development time clears current ruler marks. Measurement-notebook records remain fixed. A pigment removed from the mixture has no band to measure.

Try this in class

  1. Q1Why must the solvent-front distance be measured from the origin rather than from a pigment?
  2. Q2Why does a broadened band make its centre harder to measure?

Frequently asked questions

Can the starting Rf values identify pigments in any real solvent?

No. Rf depends on solvent and stationary phase. The reference values are explicitly illustrative.

Why can my measured Rf differ from the model Rf?

The ruler handles are intentionally independent measurement tools. Their placement can introduce measurement error.