Reversed-phase HPLC overview

A systematic overview of the principles involved in and model applications of the reverse-phase HPLC separation of carotenoids. 

Figure 5.4-1. Schematic overview of typical reversed phase-HPLC with precolumn and column loading for peptide separation. The central switching valve has two positions In position one, the sample is loaded onto the precolumn with the loading pump. After trapping and desalting of the sample, the valve is switched and the precolumn is integrated into the flow of the gradient pump. By increasing the amount of solvent B, the single peptides are separated on the separation column and afterward detected with a UV-detector and/or by mass spectrometry.

This chapter provides an overview of modern HPLC method development and discusses approaches for initial method development (column, detector, and mobile phase selection), method optimization to improve resolution, and emerging method development trends. The focus is on reversed-phase methods for quantitative analysis of small organic molecules since RPLC accounts for 60-80% of these applications. Several case studies on pharmaceutical impurity testing are presented to illustrate the method development process. For a detailed treatment of this subject and examples of other sample types, the reader is referred to the classic book on general HPLC method development by L. Snyder et al.1 and book chapters2,3 on pharmaceutical method development by H. Rasmussen et al. Other resources include computer-based training4 and training courses.5... 

As it already has been described in the overview, SDC is based on two well-polished surfaces, slid one at the top of the other generating a fluid flow thanks to the viscous drag. For chromatographic purposes, like in reverse-phase high-performance liquid chromatography (RP-HPLC), the sample solution (present in the mobile phase) needs to flow over a separation surface (the stationary phase) that can adsorb or absorb the sample... 

Fig. 5. Schematic overviews of the comprehensive on-line 2D-HPLC set-up, including size-selective sample fractionation and four parallel reversed-phase columns.

This chapter provides an overview of the UHPLC method development process and the conversion process of an HPLC method to UHPLC or vice versa. It mainly focuses on analytical reversed phase UHPLC method development of small molecules. A general process and detailed steps are discussed as well as practical... 

---