JAWAHARLAL NEHRU TECHNOLOGICAL UNIVERSITY HYDERABAD Pharm. D III Year PHARMACEUTICAL ANALYSIS Previous Question Paper (July/August - 2021 - R08)

Column efficiency in chromatography is primarily described by the Height Equivalent to a Theoretical Plate (HETP). Factors affecting it are summarized by the Van Deemter equation: H = A + B/u + Cu. The Eddy diffusion (A term) represents the multiple paths solute molecules take through the packing material; it is minimized by using uniform, small-sized spherical particles. The Longitudinal diffusion (B/u term) occurs as molecules diffuse from high to low concentration zones along the column axis; it is reduced by increasing the mobile phase flow rate. The Mass transfer resistance (Cu term) accounts for the finite time required for solute equilibrium between stationary and mobile phases; it is minimized by using thin stationary phase films and lower flow rates. Additionally, column temperature, viscosity of the mobile phase, and column packing uniformity significantly influence overall efficiency.

Paper chromatography operates on the principle of partition chromatography, where the cellulose fibers of the filter paper hold water (stationary phase) and an organic solvent (mobile phase) moves through it by capillary action. The separation depends on the differential distribution of analytes between these two immiscible phases.

Types of paper chromatography include:

• Ascending: The mobile phase moves upward against gravity.
• Descending: The mobile phase moves downward, allowing for longer runs.
• Radial (Circular): The sample is placed at the center and the solvent spreads outward.
• Two-dimensional: Used for complex mixtures; the paper is rotated 90 degrees after the first development and run in a second solvent system.

Preparation involves selecting high-quality cellulose paper, spotting the sample near the bottom edge, drying the spot, and suspending the paper in a chromatography chamber saturated with solvent vapors. Once the solvent front reaches a sufficient height, the paper is removed, dried, and the spots are visualized using specific reagents or UV light.