Unit 2: Plant Metabolism and Biochemistry Practicals

This practical aims to determine the osmotic potential of plant cell sap using the principle of plasmolysis. It utilizes the observation of cells in varying concentrations of a non-electrolyte solution like sucrose.

Principle

When a plant cell is placed in a hypertonic solution, water moves out of the cell, causing the protoplast to shrink away from the cell wall (plasmolysis). The incipient plasmolysis stage is the point where the protoplast just starts to detach from the cell wall. At this stage, the osmotic potential of the cell sap is approximately equal to the osmotic potential of the external solution.

Procedure

1. Prepare a series of sucrose solutions of known molarity (e.g., 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M).
2. Take thin epidermal peels of a plant (commonly Rhoeo discolor due to its colored sap).
3. Place one peel in each solution for about 30 minutes.
4. Examine the peels under a microscope and count the percentage of plasmolyzed cells in each concentration.
5. Determine the concentration where 50% of the cells show incipient plasmolysis. This is the isotonic concentration.

Where:
i = Ionization constant (1 for sucrose)
C = Molar concentration at incipient plasmolysis
R = Gas constant (0.0821 liter-atm/degree/mol)
T = Absolute temperature (273 + room temperature in Celsius)

This experiment investigates how the concentration of CO2, a primary substrate, affects the rate of photosynthesis in aquatic plants.

Principle

Photosynthesis uses CO2 and water in the presence of light and chlorophyll to produce glucose and oxygen. In aquatic plants like Hydrilla, the rate of photosynthesis can be measured by the rate of oxygen bubble evolution.

Experimental Setup

• An aquatic plant (Hydrilla) is placed in a funnel inverted in a beaker of water.
• A test tube filled with water is inverted over the stem of the funnel to collect evolved gas.
• Varying concentrations of CO2 are provided by adding different amounts of Sodium Bicarbonate (NaHCO3) to the water.

Observations

As the concentration of NaHCO3 increases (thereby increasing dissolved CO2), the number of bubbles produced per minute increases, up to a certain point where light or another factor becomes limiting.

Foundational to biochemical work is the accurate preparation of solutions and the use of buffers to maintain stable environments.

Types of Solutions

• Molar Solution (M): One mole of solute in 1 liter of solution.
• Molal Solution (m): One mole of solute in 1 kg of solvent.
• Normal Solution (N): One gram equivalent of solute in 1 liter of solution.
• Percent Solution (%): Grams of solute per 100 ml of solution (w/v) or ml of solute per 100 ml (v/v).

Buffers

Definition: A buffer is a solution that resists changes in pH upon the addition of small amounts of an acid or a base.

Biological processes are highly pH-sensitive. Common laboratory buffers include Phosphate buffer and Tris-HCl buffer. Preparation involves mixing a weak acid with its conjugate base (or vice versa) in specific ratios calculated using the Henderson-Hasselbalch equation.

Chromatography is a technique used to separate mixtures based on the differential distribution of components between a stationary phase and a mobile phase.

Mechanism

• Stationary Phase: The water molecules trapped in the cellulose fibers of the filter paper.
• Mobile Phase: A solvent mixture (e.g., Butanol:Acetic Acid:Water in 4:1:5 ratio).
• Amino acids travel at different rates based on their solubility in the solvent and their affinity for the paper.

Detection

Since amino acids are colorless, the paper is sprayed with Ninhydrin reagent and heated. This reacts with amino acids to produce a purple/violet color (except proline, which turns yellow).

Rf (Retention Factor) is constant for a specific substance under standard conditions and is used for identification.

This practical separates the different pigments involved in photosynthesis found in green leaves.

Pigments Separated

Significance

Separation confirms that "chlorophyll" is actually a mixture of several pigments, each absorbing light at different wavelengths to maximize photosynthetic efficiency.