Chromatography
The process involves two phases:
- Stationary phase: The material that stays fixed in place (e.g., the cellulose fibers of the chromatography paper).
- Mobile phase: The fluid that moves through the stationary phase (e.g., the solvent).
How separation occurs:
Building on the concept of solubility, components in a mixture separate because they have different affinities for the two phases:
- Components that are more soluble in the mobile phase and have less attraction to the stationary phase travel further up the paper.
- Components that are less soluble in the mobile phase and have stronger attraction to the stationary phase travel a shorter distance.
This differential movement causes the mixture to split into distinct spots or bands.
For soluble coloured substances, the components are visible as they separate. However, for soluble colourless substances (such as amino acids or sugars), a locating agent is required.
A locating agent is a chemical spray that reacts with the colourless substances to produce coloured spots, making them visible for analysis.
The equation is:
R_f = \frac{\text{distance travelled by substance}}{\text{distance travelled by solvent}}
Where:
- Distance travelled by substance: Measured from the center of the original spot (baseline) to the center of the separated spot.
- Distance travelled by solvent: Measured from the baseline to the highest point reached by the solvent (the solvent front).
Important Note: R_f values are only valid if the same solvent and paper type are used. They are not independent of solubility; a substance's R_f is determined by its solubility in that specific solvent.
- Baseline to Solvent Front: 10.0 cm
- Baseline to Spot A: 4.0 cm
- Baseline to Spot B: 8.0 cm
Calculations:
R_f \text{ (Spot A)} = \frac{4.0}{10.0} = 0.4
R_f \text{ (Spot B)} = \frac{8.0}{10.0} = 0.8
Identification:
If a known sample of 'Dye X' has an R_f value of 0.8, and Spot B also has an R_f of 0.8, then Spot B is likely Dye X.
| Interpretation |
|---|
| The substance is pure (contains only one component). |
| The substance is an impure mixture. |
| The unknown substance likely contains that specific component (same R_f value). |
The Correct Understanding: The position of a spot is determined by solubility and affinity, not by the intensity or darkness of the colour. A faint spot can travel higher than a dark spot if it is more soluble in the solvent. Do not use colour intensity to predict R_f values.
Why examiners accept this: Examiners look for the concept of solubility. Ink is soluble in many solvents and would dissolve into the mobile phase, contaminating the results. Pencil graphite is insoluble.
Correct phrasing: "The baseline is drawn in pencil because pencil marks are insoluble in the solvent. If ink were used, it would dissolve and run up the paper, interfering with the separation of the mixture."
Key term to include: Insoluble.
Why examiners accept this: If the paper is left too long, the solvent front reaches the top and evaporates, making R_f calculation impossible. If removed too early, separation may be insufficient.
Correct phrasing: "The paper should be removed when the solvent front is near the top of the paper (or just before it reaches the top). This ensures maximum separation while allowing for accurate measurement of the solvent distance."
Key term to include: Solvent front.