PRINCIPLE OF HPLC
Fig. 1 Schematic layout of a HPLC system
The separation principle of HPLC is based on the distribution of the analyte (sample) between a mobile phase (eluent) and a stationary phase (packing material of the column). Depending on the chemical structure of the analyte, the molecules are retarded while passing the stationary phase. The specific intermolecular interactions between the molecules of a sample and the packing material define their time “on-column”. Hence, different constituents of a sample are eluted at different times. Thereby, the separation of the sample ingredients is achieved. A detection unit (e.g. UV detector) recognizes the analytes after leaving the column. The signals are converted and recorded by a data management system (computer software) and then shown in a chromatogram. After passing the detector unit, the mobile phase can be subjected to additional detector units, a fraction collection unit or to the waste. In general, a HPLC system contains the following modules: a solvent reservoir, a pump, an injection valve, a column, a detector unit and a data processing unit (Fig. 1). The solvent (eluent) is delivered by the pump at high pressure and constant speed through the system. To keep the drift and noise of the detector signal as low as possible, a constant and pulseless flow from the pump is crucial. The analyte (sample) is provided to the eluent by the injection valve.
Fig. 3 an analytical and a preparative column
A. Gradient vs. isocratic
Depending on the composition of the mobile phase, two different modes are generally applicable. If the makeup of the mobile phase remains constant during the separation process, the HPLC system is defined as an isocratic elution system. When the composition of the mobile phase is changed during separation, the HPLC system is defined as a gradient elution system.Using a gradient system, two different techniques are available: a low-pressure gradient (LPG) and a high-pressure gradient (HPG). A low-pressure gradient means that the mixing of the solvents is carried out upstream of the pump (suction side). In a high-pressure gradient system, the different solvents are supplied by individual pumps and mixed after the pumps (discharge-side).
The column represents the heart of any HPLC system. It is responsible for the adequate separation of the sample ingredients. The separation efficiency correlates with the column inner diameter, the length of the column and the type and particle size of the column packing material. Depending on the desired application, numerous HPLC columns are commercially available. Different packing materials support different separation mechanisms – common are materials for normal-phase, reversed-phase, size exclusion, ion exchange, affinity, chiral, or hydrophilic interaction HPLC. In Fig. 3 an analytical and a preparative column are shown.
The task of the detector unit is to register the time and amount of a substance which is eluted from the column. The detector perceives the change in the composition of the eluent and converts this information into an electrical signal which is evaluated by the aid of a computer. A variety of detectors is available depending on the structural characteristics of the analyte. Common detector units are refractometric, UV/VIS, electrochemical and fluorescence detectors.
Fig. 4 Isocratic system configuration with a single wavelength UV-detector (left) and a refractive index detector (right)
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