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Unit 5: Case Studies

This unit applies the principles from Units 1 and 2 to specific, common laboratory separations.

Techniques for Binary Solid Mixtures (Solvent Extraction)

This technique uses the different acid-base properties of organic compounds to separate them. By changing the pH, a compound can be converted from a neutral, organic-soluble form into an ionic, water-soluble salt.

The Principle:

  1. Dissolve: The binary mixture (e.g., an acid and a neutral compound) is dissolved in an organic solvent (like ether or dichloromethane).
  2. Extract: The organic solution is shaken in a separatory funnel with an aqueous *basic* solution (e.g., NaOH(aq) or NaHCO3(aq)).
    • The acidic compound reacts with the base to form a water-soluble salt. It moves from the organic layer to the aqueous layer.
      R-COOH (Org-soluble) + NaOH → R-COO-Na+ (Water-soluble) + H2O
    • The neutral compound does not react and *stays* in the organic layer.
  3. Separate: The two layers are separated.
  4. Regenerate:
    • Aqueous Layer: Is re-acidified (e.g., with HCl). This protonates the salt, making it insoluble in water again.
      R-COO-Na+ + HCl → R-COOH(s) + NaCl
      The pure acidic solid precipitates and can be filtered off.
    • Organic Layer: Is dried (to remove traces of water) and the solvent is evaporated, leaving the pure neutral compound.

A similar logic applies for separating a basic compound (like an amine) from a neutral one, using an aqueous *acid* (e.g., HCl(aq)) for the extraction.

Case Study: Benzoic acid / p-Toluidine

This is a classic separation of an acidic compound (Benzoic acid) and a basic compound (p-Toluidine, an amine).

Procedure:

  1. Dissolve the solid mixture in an organic solvent (e.g., ether).
  2. Add dilute HCl(aq) to the separatory funnel and shake.
    • Aqueous Layer (Layer 1): The basic p-Toluidine reacts to form its water-soluble salt (R-NH3+Cl-).
    • Organic Layer (Layer A): The acidic Benzoic acid stays in the ether.
  3. Separate the layers.
  4. Treat Organic Layer (A): Add dilute NaOH(aq) and shake.
    • Aqueous Layer (Layer 2): The Benzoic acid reacts to form its water-soluble salt (R-COO-Na+).
    • Organic Layer (Layer B): *If* there was a neutral compound, it would be here. In this case, this layer is empty.
  5. Isolate the components:
    • From Layer 1: Add NaOH(aq) to make it basic. The p-Toluidine (R-NH2) precipitates and is filtered.
    • From Layer 2: Add HCl(aq) to make it acidic. The Benzoic acid (R-COOH) precipitates and is filtered.

Note: The other case studies [p-Nitrobenzoic acid (strong acid) / p-Aminobenzoic acid (amphoteric)] and [p-Nitrotoluene (neutral) / p-Anisidine (base)] are separated using the same principles.

Separation of a mixture of two amino acids by paper chromatography

  • Technique: Paper Chromatography (Partition).
  • Stationary Phase: Water molecules bound to the cellulose paper.
  • Mobile Phase (Solvent): A mixture like n-Butanol/Acetic Acid/Water.
  • Principle: Amino acids have different properties (e.g., polarity, size) due to their different "R" groups. This causes them to have different solubilities (partition coefficients) between the stationary water and the mobile organic-based solvent.
    • Procedure:
  • A pencil "origin" line is drawn near the bottom of the paper.
  • Small, concentrated spots of the amino acid mixture (and known standards) are applied to the line.
  • The paper is hung in a sealed chromatography tank with the bottom edge submerged in the solvent (below the spots).
    • Development:
  • Ascending: The solvent moves *up* the paper by capillary action.
  • Horizontal: The paper is flat, and the solvent moves *outward* from a central wick.
  • When the solvent front is near the top, the paper is removed and dried.
  • Visualization: Amino acids are colorless. The paper is sprayed with Ninhydrin solution and heated. Amino acids appear as purple/blue spots (proline gives a yellow spot).
  • Analysis: The Rf value of each spot is calculated and compared to the standards to identify the amino acids.

    • Separation of a mixture of two sugars by ascending paper chromatography

  • Technique: Paper Chromatography (Partition).
  • Principle: Identical to the amino acid separation. Different sugars (e.g., glucose, fructose, lactose) are all polar, but they have slightly different polarities and sizes, leading to different partition coefficients and thus different Rf values.
  • Procedure: Same as for amino acids (spotting, ascending development).
  • Visualization: Sugars are also colorless. They can be visualized by spraying with a reagent like Aniline Phthalate or Ammoniacal Silver Nitrate and heating, which produces colored spots.

    • Separation of o-and p-nitrophenol by Thin Layer Chromatography (TLC)

  • Technique: Thin Layer Chromatography (Adsorption).
  • Stationary Phase: Silica gel (polar) on a plate.
  • Mobile Phase: A non-polar or moderately polar solvent (e.g., Toluene or Hexane/Ethyl Acetate mixture).
  • Principle: Separation is based on polarity and adsorption.
    • p-nitrophenol (para): This molecule is very polar. It can form strong intermolecular hydrogen bonds with *other* p-nitrophenol molecules and with the polar silica gel. It will be strongly adsorbed by the stationary phase.
    • o-nitrophenol (ortho): This molecule is less polar. It forms a weak *intramolecular* hydrogen bond (between its own -OH and -NO2 groups). This "internal" H-bond prevents it from H-bonding strongly with the silica gel.
  • Result:
    • The o-nitrophenol (less polar, weakly adsorbed) travels *quickly* up the plate with the solvent. It will have a high Rf value.
    • The p-nitrophenol (more polar, strongly adsorbed) "sticks" to the origin. It will have a very low Rf value.
  • Visualization: Both compounds are yellow, so they can be seen directly.
  • Note: The same principle applies to separating o- and p-aminophenol.
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