Basic Information about HPLC

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1.1. INTRODUCTION TO HPLC What is Chromatography? The term chromatography designates several similar techniques that allow the separation of different molecular species from a mixture. Applications of chromatography are numerous and can be related to laboratory or industrial practices. The molecular species subjected to separation exist in a sample that is made of analytes and matrix. The analytes are the molecular species of interest, and the matrix is the rest of the components in the sample.

For chromatographic separation, the sample is introduced in a flowing mobile phase that passes a stationary phase. The stationary phase retains stronger or weaker different passing molecular species and releases them separately in time, back into the mobile phase. When the mobile phase is a gas, the chromatography is indicated as gas chromatography (GC), and when it is a liquid, it is indicated as liquid chromatography (LC). Other types of chromatography include supercritical fluid, countercurrent, and electrochromatography. When the sample is present as a solution, its components are indicated as solutes. Sample dissolution and/or preliminary modifications are frequently necessary to have the analytes amenable for a chromatographic separation (see, e.g., [1]).

In high performance (or pressure) liquid chromatography (HPLC), the stationary phase is typically in the form of a column packed with very small porous particles (1e5 mm in diameter), and the liquid mobile phase (or eluent) is moved through the column by a pump (at elevated pressure). Solutes are injected in the mobile phase as a small volume at the head of the chromatographic column. A schematic diagram of the separation process is shown in Figure 1.1.1. As the mobile phase flows, the eluted molecules that are exiting the column can be detected by various procedures. The eluted molecules differ from the mobile phase components by certain physicochemical properties (UVabsorption, refractive index, fluorescence, molecular mass and fragmentation in a mass spectrometer, or others), which make them detectable. Finally, an electrical signal is typically associated with molecular detection, and the graphic output of this signal is known as a chromatogram. The separated components of a mixture eluting at different times (known as retention times tR) are displayed as peaks in the chromatogram. Different peaks (or patterns) on the chromatogram belong to different components of the separated mixture. An example of a chromatogram with the retention times written above the peaks is shown in Figure 1.1.2. As shown in this figure, the separation of the peaks can be very good or only partial. Also, some compounds may not be separated at all. Separated peaks may indicate individual compounds only when each peak corresponds to a single molecular species.