The analyte (sample) is provided to the eluent by the injection valve. 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 solvent (eluent) is delivered by the pump at high pressure and constant speed through the system. 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. After passing the detector unit, the mobile phase can be subjected to additional detector units, a fraction collection unit or to the waste. This approach uses two chambers, where one chamber contains the acrylamide solution at the lowest gradient concentration, and the other contains the higher acrylamide concentration (Fig. The signals are converted and recorded by a data management system (computer software) and then shown in a chromatogram. The same rules apply when making a gradient gel. UV detector) recognizes the analytes after leaving the column. Thereby, the separation of the sample ingredients is achieved.Ī detection unit (e.g. Hence, different constituents of a sample are eluted at different times. The specific intermolecular interactions between the molecules of a sample and the packing material define their time “on-column”. Depending on the chemical structure of the analyte, the molecules are retarded while passing the stationary phase. 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).
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