Questions
2 questions per paper
Difficulty
Medium
Importance
Core topic for B.Pharm and BMLT/BMRIT university exams
Overview
Chromatography is a versatile laboratory technique used for the separation, identification, and purification of components in a mixture based on differential partitioning between stationary and mobile phases. In academic exams, understanding the polarity principles and retention mechanisms is critical, as it forms the backbone of analytical chemistry in pharmacy and clinical sciences.
Thin Layer Chromatography (TLC)
TLC is a simple, rapid technique utilizing a thin layer of adsorbent material coated on a flat, inert substrate. Separation occurs via capillary action as the mobile phase moves over the stationary phase, differentiating components by their relative affinity.
- Stationary phase: Silica gel or Alumina
- Retardation factor (Rf) = Distance traveled by solute / Distance traveled by solvent front
- Visualization: UV lamp, Iodine vapors, or chemical spray reagents
- Ideal for qualitative analysis and reaction monitoring
High-Performance Liquid Chromatography (HPLC)
HPLC is a highly automated and sensitive analytical method that uses high pressure to push the solvent through a densely packed column. It is the gold standard for quantitative analysis in the pharmaceutical industry for drug assay and impurity profiling.
- Normal phase: Polar stationary phase, non-polar mobile phase
- Reverse phase: Non-polar stationary phase (C18), polar mobile phase
- Detector types: UV-Vis, PDA, Fluorescence, or Refractive Index
- Separation based on hydrophobicity and solubility
Gas Chromatography (GC)
Gas chromatography is designed for the separation of volatile and thermally stable compounds. The analyte is vaporized and carried through a capillary column by an inert carrier gas such as Helium, Nitrogen, or Hydrogen.
- Analyte must be volatile and thermally stable
- Common detectors: Flame Ionization Detector (FID) and Thermal Conductivity Detector (TCD)
- Stationary phase is a high-boiling point polymer liquid coated on the inner wall of the capillary
- Separation based on boiling point and partition coefficient
Formula Sheet
Rf = (Distance of solute) / (Distance of solvent front)
Resolution (Rs) = 2 * (tR2 - tR1) / (w1 + w2)
Theoretical plates (N) = 16 * (tR / w)^2
Exam Tip
Always draw a labeled diagram for the instrumentation of HPLC or GC, as these diagrams typically carry 30-40% of the marks in long-answer questions.
Common Mistakes
- Confusing the roles of stationary and mobile phases in reverse-phase HPLC.
- Forgetting that the Rf value is a unitless ratio and must always be less than 1.
- Assuming GC can be used for non-volatile biological macromolecules without prior derivatization.
More Revision Notes
Ready to test yourself?
Play topic-wise Chromatography questions in Aspirant Arcade — gamified MCQ practice.
Download Free