Overview of Separation Methods

Whereas individual components to be quantitated may be isolated to meet the 1.5% criteria outlined and subsequently quantitated, the more common approach is the use of separation procedures, coupled to suitable detection, to separate and monitor the individual analytes of interest. This is a particularly powerful approach because several of the requisite quantitative measurements can be made from a single analysis of the sample. Using optimized separation procedures, it is possible to monitor the API (for assay), organic synthetic process impurities, and degradation products during a single determination. Similarly, multiple residual solvents may be quantitated in a single analysis. 

Chemical separations are achieved using chromatographic and, to a much lesser extent, electrophoretic methods. In chromatographic methods, separation is based on variation in the distribution of different compounds between two dissimilar phases—a stationary phase and a mobile phase. Further differentiation can be made between chromatographic procedures in which the individual components elute from the stationary phase and are monitored on line (column chromatography) and procedures in which the components are measured in situ on the chromatographic stationary phase [e.g., thin-layer 

The primary goal of chromatographic separations is to separate the components (of interest) in the sample. The condition of separation is achieved when the resolution (Rs) between individual components is greater than a numerical value of 2.0. Resolution can be measured from a chromatogram as detailed in the USP. However, the measurement of resolution does not direct the approach to achieving resolution. 

All column chromatographic separations operate on the same principle the distribution of solute between two dissimilar phases—a mobile phase and a stationary phase. In each procedure, sample is introduced into one end of the column and the mobile phase transports the sample components toward the other end of the column. In the absence of interaction with the stationary phase, all components would exit the other end of the column after a time, to, based on the column volume and mobile-phase flow rate and could be detected at the other end of the column using an on-line detector. If an interaction with the stationary phase occurs, the time taken for a solute to elute from the column would be increased by the time that the solute spends in association with the stationary phase (tj ). The ratio of is directly proportional to the distribution coefficient between the stationary phase and the mobile phase and is referred to as the capacity factor (or retention factor) k. The ratio of the k s of two solutes to be separated, where the subscripts denote the second 

Resolution, previously described as the degree of separation between components, can be expressed in terms of k, a, and N as 

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F. Tagliaro, D. Franchi, R. Dorizzi and M. Marigo, High-performance liquid chromatographic determination of morphine in biological samples an overview of separation methods and detection techniques, J. Chromatogr., 1989, 488, 215-228. 

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