Hplc System Stability
HPLC Theory: System Suitability Parameters High performance liquid chromatography is defined as a separation of mixtures of compounds due to differences in their distribution equilibrium between two phases, the stationary phase packed inside columns and the mobile phase, delivered through the columns by high pressure pumps. Components whose distribution into the stationary phase is higher, are retained longer, and get separated from those with lower distribution into the stationary phase. The theoretical and practical foundations of this method were laid down at the end of 1960s and at the beginning of 1970s. The theory of chromatography has been used as the basis for System- Suitability tests, which are set of quantitative criteria that test the suitability of the chromatographic system to identify and quantify drug related samples by HPLC at any step of the pharmaceutical analysis. Retention Time (tR), Capacity Factor k ' and Relative Retention Time (RRT) The time elapsed between the injection of the sample components into the column and their detection is known as the Retention Time (tR). The retention time is longer when the solute has higher affinity to the stationary phase due to its chemical nature. For example, in reverse phase chromatography, the more lypophilic compounds are retained longer. Therefore, the retention time is a property of the analyte that can be used for its identification. A non retained substance passes through the column at a time t0, called the Void Time. The Retention Factor or Capacity Factor k ' of an analyte is measured experimentally as shown in Figure 3 and Eqn 1: Eqn 1a [pic] The Capacity Factor describes the thermodynamic basis of the separation and its definition is the ratio of the amounts of the solute at the stationary and mobile phases within the analyte band inside the chromatographic column:
Eqn 1b [pic] Where Cs is the
Eqn 1b [pic] Where Cs is the
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