3.3.2 High-Performance Thin-Layer Chromatography (HPTLC) Fingerprinting

High-performance thin-layer chromatography extends conventional TLC through instrumentation that automates and standardises every step of the process —...

High-performance thin-layer chromatography extends conventional TLC through instrumentation that automates and standardises every step of the process — precise, automated sample application (commonly by a device such as the Linomat applicator), controlled plate development in a twin-trough chamber, and densitometric scanning of the developed plate under multiple wavelengths and illumination modes — yielding a fully quantitative chromatographic fingerprint rather than a purely visual pattern. Class-specific derivatising agents are again employed, including anisaldehyde-sulphuric acid (producing characteristic blue, purple, or pink colouration for terpenoids and saponins), Dragendorff's reagent (orange bands for alkaloids), and natural product/polyethylene glycol reagent (yellow-orange fluorescence for flavonoids under UV 366 nm). Validated HPTLC fingerprinting demands high reproducibility, with intra-day retention factor variation conventionally required to remain within one percent relative standard deviation and inter-day variation within two percent, and the technique is now formally recognised by the Indian Pharmacopoeia as a mandatory identification test for herbal raw materials and extracts.