ANALYSIS OF SAMPLE BY HIGH PERFORMANCE THINLAYER CHROMATOGRAPHY (HPTLC) 13, 14
High performance thin-layer chromatography (also known as planar or flatbed chromatography) is basically an enhanced form of Thin-Layer Chromatography (TLC). Several enhancements can be made to conventional method of TLC for different reasons namely automation of desired steps, for higher resolution and to achieve more accurate quantitative measurement.
a) Principle: HPTLC follows the exact same principle as that of TLC i.e., separation of components is based on adsorption. The sample components get separated based on their affinities towards the stationary phase (adsorbent) as well as their solubility in the mobile phase (solvent). The solvent containing the sample is made to flow through the adsorbent layer (by capillary action), the components with higher affinity travels slower compared to the ones with lower affinity that travel faster.
Fig.5: Instruments involved in HPTLC.
b) Working: The steps involved in analysis by HPTLC are as follows
? Chromatographic plate selection
? Layer pre-washing
? Activation of pre-coated plates
? Preparation of the sample and its application
? Mobile phase selection
? Development of chromatographic plates and its drying
? Detection of sample spots and their visualization ? Documentation of results.
c) Applications of HPTLC:
• In pharmaceutical industries for qualitative and quantitative tests
• For food analysis
• In clinical studies like drug screening, stability testing etc
• Forensic investigation.
d) Specifications for sample analysis by TLC:
? Mobile phase : toluene : ethyl acetate : methanol (7:2:1)
? Stationary phase : Silica gel-G
? Detection system : UV 366nm
e) Specifications for sample analysis by HPTLC:
? Made / make of instrument :DesagaSarstedt Gruppe (Germany)
? Development chamber : 20 x 10cm, Twin-trough chamber
? Stationary phase : pre coated silica gel 60 F254 Aluminium plates (Merck, KgaA, Germany)
? Plate thickness : 0.2mm
? Plate size : 200 x 100mm
? Distance from starting : 20mm
? Distance from bottom : 10mm
? Volume applied : 5µl
? Band length : 10mm
? Distance between tracks : 20mm
? Development distance : 80mm
? Solvent used : HPLC grade
? Extract storage vials : 5ml glass vials
? Software : Proquant 1.6 version
? Mobile phase / solvent system : toluene : ethyl acetate : methanol (7:2:1)