High-resolution polymer high performance liquid chromatography: optimization of the saw tooth gradient profile for various stationary phases and separations on preparative scale

Abstract

The recently introduced saw tooth gradient protocol for high-resolution polymer HPLC was further improved and optimized in terms of total runtime and separation performance. As a result, increased flow rates enabled drastically reduced runtimes in combination with enhanced peak resolutions. Moreover, the saw tooth gradient profile was further investigated using a saw tooth gradient with a down-to-zero approach concerning the height of the negative backward gradient step. Modifying the mobile phase composition enabled two further gradient protocols: a ternary, and a multicomponent approach, respectively. Thereby, a ternary saw tooth gradient was realized by repeating the whole gradient elution with two adequate pairs of weak or rather non-solvents and desorption promoting solvents for mixtures containing diverse polymer components. A multicomponent saw tooth gradient was established by combining three different solvents in the gradient elution. In addition to mobile phase modifications, various stationary phases were compared and examined. Applying size exclusion chromatography (SEC) columns for saw tooth gradient polymer elution chromatography enabled the exploitation of two completely different separation mechanisms (SEC and high-resolution polymer HPLC) on one stationary phase. Thus, two-dimensional, heart-cut coupling of SEC and high-resolution polymer HPLC with only one stationary phase could be achieved. The application of the above-mentioned concept and its highly attractive performance characteristics are demonstrated for a silicone oil with a viscosity of 350 mPa s by using a hybrid HPLC system coupled to a fraction collector.