Columns, reversed HPLC fundamentals

A variety of micropellicular packing materials has been developed for the analysis of both small and large molecules by various HPLC modes, including ion exchange (lEC), metal interaction (MIC), reversed-phase (RPC), and affinity chromatography (AC). Besides analytical applications, other possible utilization of micro-pelhcular stationary phases includes fundamental kinetic and thermodynamic studies of the retention mechanisms on a well-defined surface. Nevertheless, a relatively limited variety of micropellicular columns are commercially available. They are mainly restricted to ion-exchange and reversed-phase stationary phases. This may reflect certain practical disadvantages of micropelhcular sorbents. 

The fundamental components of any modern-day HPLC system are a solvent delivery system, a sample injector, a column, a detector, and a computer with the appropriate data acquisition and processing software. There are numerous HPLC methods described in the literature for isoflavones [13-25] and for the common anthocyanins, each method invokes different combinations of solvent systems, columns, and detectors. HPLC has been interfaced with a variety of detection methods such as ultraviolet/visible (UV/vis) spectrocopy and hquid chromatography-mass spectrometry (LC-MS) [21,22]. In this chapter, however, discussion is restricted to the most commonly used pairing in flavonoid analysis, that of a reverse-phase (RP-18) column and a UV/visible detector. 

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