Preparation: A horizontal line is drawn in pencil near the bottom of the chromatography paper to mark the starting point (origin).
Spotting: Small, concentrated drops of the unknown mixture and known standard solutions are placed on the pencil line and allowed to dry.
Development: The paper is suspended in a container with a solvent, ensuring the solvent level is below the pencil line to prevent the samples from dissolving directly into the bulk solvent.
Termination: The process is stopped before the solvent reaches the top of the paper, and the highest point reached by the solvent (the solvent front) is immediately marked.
Because amino acids are naturally colorless, they must be visualized using a chemical developer such as ninhydrin solution.
When sprayed with ninhydrin and dried, the amino acids undergo a chemical reaction that produces a visible blue-violet color.
Identification is achieved by matching the distance traveled by the unknown spots with the distances traveled by the known standard spots.
If an unknown spot travels the exact same distance as a known standard under identical conditions, it is highly likely that the unknown contains that specific amino acid.
| Feature | Mobile Phase | Stationary Phase |
|---|---|---|
| Physical State | Liquid solvent | Solid (cellulose paper) |
| Movement | Moves upward via capillary action | Remains fixed in the container |
| Solute Interaction | Carries highly soluble solutes further | Retards the movement of less soluble solutes |
| Function | The transport medium | The separation medium |
Pencil vs. Ink: Always remember that the origin line must be drawn in pencil; ink contains dyes that would dissolve in the solvent and interfere with the results.
Solvent Level: Ensure the solvent starts below the sample spots; if the spots are submerged, they will wash off into the solvent rather than traveling up the paper.
Standard Comparison: In exam questions, look for spots that align horizontally; identical vertical displacement indicates identical chemical identity.
Visualization: If a question asks how to see 'invisible' spots, the answer is almost always the application of ninhydrin spray.
Submerging the spots: A common error is adding too much solvent, which causes the amino acids to diffuse into the beaker rather than separating on the paper.
Confusing solubility with speed: While more soluble components travel further, this is because they spend more time in the mobile phase, not necessarily because they 'swim' faster.
Ignoring the solvent front: Failing to mark the solvent front immediately after removal can make it impossible to calculate relative migration if the solvent evaporates.
