Fast Analysis of Bioactive Compounds by Reverse Phase Liquid Chromatography

Abstract

The analysis of bioactive compounds in raw bioactive materials by liquid chromatography can prove to be a very difficult task and usually require extended times to adequately separate all components from such complex matrices. Currently there is a great interest in the development of new technology to improve chromatographic performance and reduce the necessary time to achieve the separation between components. Liquid chromatography has seen great improvements in the last decades, especially in stationary phase technology. New stationary phase technologies include sub2 µm particles, partially porous and porous monolithic polymers. These new packing materials are taking the chromatographic performance of separations to a much higher level. Liquid chromatography systems are also seen improvements in term of pressure limits, precision, reproducibility and overall quality of the results. The use of these advanced systems and materials allied to the adoption of carefully planned strategies under optimized conditions can produce excellent chromatographic results with speeds that were thought to be impossible a few years ago. In this context, this chapter discusses current technology available that can be explored to reduce analysis time of bioactive compounds in natural products. It also discusses the influence of most important operational parameters (column In Instrumental Methods for the Analysis and Identification of Bioactive Molecules; Jayprakasha, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2014.temperature, mobile phase composition and flow rate/ linear velocity in the chromatographic system and how these variables can be adjusted in order to produce the greatest potential for performing separations.

Liquid Chromatographic Strategies for Separation of Bioactive Compounds in Food Matrices

“…Due to the complexity of food matrices and the great structural variety of the substances they contain, the separation of bioactive compounds and their analysis by HPLC is not an easy task, often requiring long run times. Nevertheless, in the last decades, as for other fields, research on bioactive compounds is obtaining advantages by the development of new HPLC stationary phase technologies, including sub-2 µm particles, partially porous particles, and porous monolithic polymers [12].…”

“…The employment of smaller-particle chromatographic packing materials allows improvement of resolution, efficiency, and method sensitivity, because the resulting chromatographic peaks are sharper and higher [12]. The high resulting backpressure can be alleviated by using short columns, which also guarantee low dead volumes.…”

“…All these stationary phase technologies have advantages and drawbacks, and for deepening these topics, the reader can refer to the specific literature [14,15,16,17,18]. Nonetheless, separation efficiency is not only dependent on stationary phase typology, but it is strongly affected by the optimization of operational parameters, such as column temperature, mobile phase composition, and flow-rate [12]. In particular, because temperature affects both the kinetics and thermodynamics of the chromatographic process, operating at temperatures > 30 °C reduces solvent viscosity, leading to a lower column backpressure which can be explored to increase mobile phase flow-rate and speed up LC separations [12].…”