Principle of HPLC
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.
Delay time (t0)
The delay time refers to the time which is required for a non-retarded compound to be transported from the injection site to the detector unit (where the compound is recorded). During this time, all sample molecules are exclusively located in the mobile phase. In general, all sample molecules share the same delay time. The separation is caused by differing adherence of the substances with the stationary phase.
Retention time (tR)
The retention time refers to the time which is required for a compound from the moment of injection until the moment of detection. Accordingly, it represents the time the analyte is in the mobile and stationary phase. The retention time is substance-specific and should always provide the same values under the same conditions.
Peak width (w)
The peak width covers the period from the beginning of the signal slope until reaching the baseline after repeated drop in the detector signal.