Class 12 Chemistry Notes Chapter 5 (Chromatography) – Lab Manual (English) Book

Detailed Notes with MCQs of a very important separation technique you've encountered in the lab: Chromatography. This technique is fundamental not just for your practical exams but also frequently appears in various government competitive exams due to its wide applicability. We'll break down the key concepts based on your NCERT Lab Manual.

Chapter 5: Chromatography - Detailed Notes

1. Introduction

• Definition: Chromatography is a powerful laboratory technique used for the separation, identification, and purification of components in a mixture.
• Origin: The term comes from Greek words 'chroma' (colour) and 'graphein' (to write), as the technique was first used by the Russian botanist Mikhail Tsvet in 1906 to separate coloured plant pigments.
• Basis of Separation: The technique relies on the differential distribution or partitioning of the components of a mixture between two phases: a stationary phase and a mobile phase.

2. Principle of Chromatography

• The core principle is the differential movement of individual components of a mixture through a stationary phase under the influence of a mobile phase.
• Components that are more strongly retained by the stationary phase move slower.
• Components that are less strongly retained by the stationary phase (or are more soluble in the mobile phase) move faster.
• This difference in movement leads to the separation of the components.
• The distribution can be based on various mechanisms like adsorption, partition, ion exchange, size exclusion, etc. Your lab manual primarily focuses on adsorption and partition, especially in the context of paper and thin-layer chromatography.

3. Key Terms

• Stationary Phase: A solid or a liquid supported on a solid, which remains fixed in place. It selectively retains components of the mixture.
  • Examples (from lab manual context): Silica gel or Alumina (in TLC), Water adsorbed on cellulose paper (in Paper Chromatography).
• Mobile Phase: A liquid or a gas that flows over or through the stationary phase, carrying the components of the mixture along with it.
  • Examples (from lab manual context): Organic solvents or mixtures of solvents (like ethanol, methanol, water, acetone, petroleum ether, etc.).
• Adsorption: The process where components of the mixture adhere to the surface of the solid stationary phase.
• Partition: The process where components distribute themselves between two immiscible liquid phases (one being the stationary liquid phase, often adsorbed on a solid support, and the other being the mobile liquid phase).
• Chromatogram: The visual output of chromatography. In paper or TLC, it's the developed paper or plate showing separated spots.
• Developing Solvent: Another name for the mobile phase, specifically the liquid used to carry the mixture components up the stationary phase.
• Spotting: The process of applying a small, concentrated spot of the mixture onto the starting line (origin) of the chromatogram (paper or plate).
• Solvent Front: The furthest point reached by the mobile phase on the chromatogram.

4. Types of Chromatography (Focus on Lab Manual Experiments)

• A. Paper Chromatography:

  • Principle: Primarily partition chromatography, although adsorption on cellulose also plays a role.
  • Stationary Phase: Water molecules trapped/adsorbed in the pores of the cellulose fibres of the chromatography paper (special paper like Whatman No. 1). The paper itself acts as the support.
  • Mobile Phase: A suitable solvent or mixture of solvents (e.g., water, ethanol, acetic acid mixture).
  • Procedure Highlights:
    1. Cut the paper strip; draw a starting line (origin) with a pencil.
    2. Apply a small, concentrated spot of the mixture on the origin.
    3. Suspend the paper in a closed chromatography jar/chamber containing the mobile phase. Ensure the spot is above the solvent level.
    4. Allow the solvent to rise up the paper by capillary action (development).
    5. When the solvent front nears the top, remove the paper and mark the solvent front.
    6. Dry the paper.
    7. Visualize the spots (if colourless, use a locating/visualizing agent like Ninhydrin for amino acids, or specific reagents for inorganic ions).
  • Applications (Lab Manual): Separation of plant pigments (chlorophylls, carotenoids), separation of coloured inks, separation of inorganic cations (like Pb²⁺ and Cd²⁺, Ni²⁺ and Co²⁺), separation of amino acids.

• B. Thin Layer Chromatography (TLC): (Often discussed as an alternative/improvement)

  • Principle: Primarily adsorption chromatography.
  • Stationary Phase: A thin layer (e.g., ~0.25 mm) of an adsorbent like silica gel (SiO₂) or alumina (Al₂O₃) coated uniformly on a glass plate, plastic sheet, or aluminium foil.
  • Mobile Phase: A suitable solvent or mixture of solvents.
  • Advantages over Paper Chromatography: Faster separation, better resolution (sharper spots), wider choice of adsorbents.
  • Procedure: Similar to paper chromatography (spotting, development in a chamber, drying, visualization).

5. Retention Factor (Rf Value)

• Definition: The ratio of the distance travelled by a component (solute) from the origin to the distance travelled by the mobile phase (solvent front) from the origin.
• Formula:
  Rf = (Distance travelled by the solute from the origin) / (Distance travelled by the solvent front from the origin)
• Characteristics:
  • Rf value is always less than 1.
  • It is a characteristic constant for a given substance, on a specific stationary phase, with a specific mobile phase, and at a constant temperature.
  • It helps in the identification of components by comparing their Rf values with known standards run under identical conditions.
  • Factors affecting Rf value: Nature of adsorbent, mobile phase composition, temperature, saturation of the chamber, quality of paper/plate.

6. Applications of Chromatography (General)

• Separation of mixtures into pure components.
• Purification of compounds.
• Qualitative analysis (identification of substances based on Rf values or retention times).
• Quantitative analysis (determining the amount of each component).
• Widely used in pharmaceutical industry, forensic science, environmental analysis, food industry, clinical diagnostics, and biochemical research.

7. Precautions in Paper/TLC Chromatography

• Use high-quality chromatography paper/plates.
• Draw the origin line with a pencil (ink will separate).
• Apply the spot small and concentrated; allow it to dry completely.
• The spot must be above the solvent level in the chamber.
• The chromatography chamber must be saturated with solvent vapour for better separation and reproducible Rf values.
• Avoid disturbing the chamber during development.
• Mark the solvent front immediately after removing the chromatogram.
• Handle chromatograms carefully, especially if visualizing agents are used.

Multiple Choice Questions (MCQs)

1. Chromatography is a technique based on the principle of:
   a) Differential boiling points of components.
   b) Differential solubility of components in a single solvent.
   c) Differential distribution of components between a stationary and a mobile phase.
   d) Differential reaction rates of components.
   Answer: c)

2. In paper chromatography, the stationary phase is typically:
   a) The cellulose paper itself.
   b) Water adsorbed on the cellulose paper.
   c) The developing solvent.
   d) Silica gel coated on paper.
   Answer: b)

3. The Retention Factor (Rf) value in chromatography is defined as:
   a) (Distance travelled by solvent front) / (Distance travelled by solute)
   b) (Distance travelled by solute) / (Distance travelled by solvent front)
   c) (Distance travelled by solute) x (Distance travelled by solvent front)
   d) Distance travelled by the solute only.
   Answer: b)

4. Which of the following factors generally does NOT affect the Rf value in paper chromatography?
   a) Temperature
   b) Nature of the mobile phase
   c) Diameter of the chromatography jar
   d) Nature of the stationary phase (paper quality)
   Answer: c)

5. If two components A and B are separated by chromatography, and component A has an Rf value of 0.4 while component B has an Rf value of 0.7, which component moved faster?
   a) Component A
   b) Component B
   c) Both moved at the same speed
   d) Cannot be determined from Rf values
   Answer: b) (Higher Rf means it travelled further relative to the solvent front, hence faster)

6. In the separation of plant pigments from spinach extract using paper chromatography, the different coloured bands are formed because pigments have different:
   a) Molecular weights
   b) Densities
   c) Affinities towards the stationary and mobile phases
   d) Melting points
   Answer: c)

7. To ensure good separation and reproducible Rf values in paper or TLC, the chromatography chamber should be:
   a) Left open to allow solvent evaporation.
   b) Filled completely with the mobile phase.
   c) Kept in bright sunlight.
   d) Properly sealed and saturated with solvent vapour.
   Answer: d)

8. In Thin Layer Chromatography (TLC), a common adsorbent used as the stationary phase is:
   a) Filter paper
   b) Water
   c) Silica gel
   d) Ethanol
   Answer: c)

9. Why is the starting line drawn with a pencil on the chromatography paper/plate?
   a) Pencil lead (graphite) is inert and does not dissolve in the mobile phase.
   b) Pencil marks are easier to see.
   c) Ink contains multiple components that would separate along with the sample.
   d) Both a) and c).
   Answer: d)

10. A spot of mixture is applied to a chromatography paper and developed. The solvent front moves 10 cm. A component 'X' moves 5 cm from the origin. What is the Rf value of component 'X'?
    a) 2.0
    b) 0.5
    c) 5.0
    d) 10.0
    Answer: b) (Rf = 5 cm / 10 cm = 0.5)

Remember to relate these principles directly to the experiments you performed, like separating leaf pigments or inorganic ions. Understanding the 'why' behind each step is crucial for both lab work and exams. Good luck with your preparation!