Pharmaceutical Analysis · Short notes

Chromatography: Short Notes & Cheat Sheet

Separation principles, TLC and Rf, HPLC modes and detectors, system suitability, the van Deemter equation and gas chromatography.

Short notes

Read these before you attempt the topic's MCQs.

Retention time (from the column) identifies a compound; the detector's peak area measures how much there is.

Chromatography separates components by their different distribution between a stationary phase and a mobile phase.
Mechanisms: adsorption (TLC on silica, column), partition (paper, GC, reverse-phase HPLC), ion exchange, size exclusion (large molecules elute first) and affinity.
Rf = distance moved by the spot ÷ distance moved by the solvent front. It always lies between 0 and 1.
TLC plates use silica gel G (with a gypsum binder); F254 plates contain a fluorescent indicator so spots show as dark areas under 254 nm UV light.
Spots are also shown with iodine vapour, ninhydrin (amino acids) or Dragendorff's reagent (alkaloids).

Reverse-phase HPLC is the most common: a non-polar C18 column with a polar mobile phase (water with methanol or acetonitrile). Polar compounds elute first.
Normal-phase HPLC uses a polar stationary phase (silica) and a non-polar mobile phase.
Isocratic elution keeps the mobile phase constant; gradient elution changes its composition during the run.
Detectors: UV (most common), photodiode array, fluorescence, refractive index (universal but cannot be used with gradients), electrochemical and mass spectrometry.
Retention time identifies a compound; peak area (or height) measures how much is present.

System suitability typically requires: resolution ≥ 1.5 (often ≥ 2), tailing factor ≤ 2, enough theoretical plates, and RSD ≤ 2% for replicate injections.
Plate number N = 16 (tR/W)². Capacity factor k′ = (tR − t₀)/t₀.
Van Deemter equation: H = A + B/u + C·u (eddy diffusion, longitudinal diffusion and mass transfer). Smaller particles give better efficiency but higher back-pressure (UPLC uses particles under 2 µm).
Gas chromatography needs volatile, heat-stable analytes and a carrier gas (helium, nitrogen or hydrogen).
GC detectors: FID (most organic compounds), ECD (halogenated compounds such as pesticides), TCD (universal). Headspace GC measures residual solvents.

High-yield facts in one place. Cover the right-hand column and test yourself.

Detector	Best for
UV / PDA (HPLC)	Compounds with chromophores
Refractive index (HPLC)	Sugars; universal, isocratic only
Fluorescence (HPLC)	Trace fluorescent compounds
FID (GC)	Organic compounds
ECD (GC)	Halogenated compounds, pesticides
TCD (GC)	Universal, including gases
Mass spectrometer	Identity and trace levels

Reverse phase: Polar Passes first: On a non-polar C18 column, polar compounds are retained least and elute first.

In size-exclusion chromatography the largest molecules elute first.
A refractive index detector cannot be used with gradient elution.
Rf can never be greater than 1.
In GC the mobile phase is an inert gas that does not interact with the analytes.

Attempt the Chromatography MCQs while the notes are fresh. Anything you get wrong is saved to your incorrect list for the next revision round.