Fundamentals and Theory of HPTLC-Based Separation

Abstract

High-performance thin-layer chromatography (HPTLC) is a form of thin-layer chromatography (TLC) that provides superior separation power using optimized coating material, novel procedures for mobile-phase feeding, layer conditioning, and improved sample application. It promotes for higher separation efficiencies, shorter analysis time, lower amounts of mobile phase, and efficient data acquisition and processing. The major parameters that influence separation of the constituents within a mixture are the partition coefficients, retention factor (R f ), and capacity factor of the individual constituents on the plate, selectivity of the mobile and stationary phase to the solutes, and the plate height that decide the separation efficiency as well as resolution of the individual constituents within a mixture. The partition coefficient is defined as the molar concentration of the analyte in the stationary phase to that in the mobile phase. R f , a fundamental qualitative value, is expressed as the ratio of migration distances of an individual components of a mix relative to the mobile phase. Capacity factor (k) is a fundamental characteristic of a substance that determines its qualitative chromatographic behavior. It can be expressed as the ratio of the retention time of the substance in the stationary to that in the mobile phase and is influenced by the chemical nature of the two phases. The separation number (SN) that influences separation power of HPLC is defined as the highest possible number of components that are completely separated in a mixture under a gradient-free isocratic TLC. The efficacy of separation of two components of a mixture in a chromatogram is termed as resolution and is influenced by the selectivity of the components between the stationary and the mobile phase, mobile phase flow rate influenced by particle size and solvent strength that influence capacity factors.