Stationary phases in reversed phase HPLC

In HPLC, a sample is separated into its components based on the interaction and partitioning of the different components of the sample between the liquid mobile phase and the stationary phase. In reversed phase HPLC, water is the primary solvent and a variety of organic solvents and modifiers are employed to change the selectivity of the separation. For ionizable components pH can play an important role in the separation. In addition, column temperature can effect the separation of some compounds. Quantitation of the interested components is achieved via comparison with an internal or external reference standard. Other standardization methods (normalization or 100% standardization) are of less importance in pharmaceutical quality control. External standards are analyzed on separate chromatograms from that of the sample while internal standards are added to the sample and thus appear on the same chromatogram. 

Compared to GC columns, HPLC columns are short and thick, ranging from 10 to 25 cm in length and 2 to 4.6 mm in internal diameter. They are filled with an inert material (silica, polymer resin), which is coated with a stationary phase. In normal phase HPLC, the mobile phase is less polar than the stationary phase. In reverse phase HPLC, the opposite is true, and the mobile phase is more polar than the stationary one. Reverse phase HPLC is the technique of choice for environmental applications. Similar to GC columns, analyte-specific HPLC columns are recommended in the published methods. 

In a binary eluent system (acetonitrile-water), an adsorbed organic phase with finite thickness and composition different from the bulk mobile phase is preferentially accumulated near the surface of the bonded phase. The organic layer accumulated near the bonded ligands could behave as a liquid stationary phase in reversed-phase HPLC, and it contributes to the overall analyte retention process. 

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