High-performance liquid chromatography normal/reversed phase modes

Thick-layer silica gel chromatography can also be employed [7], although most separations are now accomplished by high-performance liquid chromatography. Resolution of complex mixtures often requires both normal and reverse phase modes [19]. A further dimension is added, when bioactivity is correlated with spectroscopically-monitored chromatographic profiles. 

High-performance liquid chromatography (HPLC) techniques are widely used for separation of phenolic compounds. Both reverse- and normal-phase HPLC methods have been used to separate and quantify PAs but have enjoyed only limited success. In reverse-phase HPLC, PAs smaller than trimers are well separated, while higher oligomers and polymers are co-eluted as a broad unresolved peak [8,13,37]. For our reverse-phase analyses, HPLC separation was achieved using a reverse phase. Cl8, 5 (Jtm 4.6 X 250 mm column (J. T. Baker, http //www.mallbaker.com/). Samples were eluted with a water/acetonitrile gradient, 95 5 to 30 70 in 65 min, at a flow rate of 0.8 mL/min. The water was adjusted with acetic acid to a final concentration of 0.1%. All mass spectra were acquired using a Bruker Esquire LC-MS equipped with an electrospray ionization source in the positive mode. 

Normal-phase liquid chromatography (NPLC) is the oldest chromatographic mode, discovered by M.S. Tswett more than 100 years ago. It has been the predominant mode in thin-layer chromatography (TLC) and low-pressure dry-column liquid chromatography before the introduction of reversed-phase technique, which is a preferred mode in high-performance liquid chromatography (HPLC). 

The normal-phase (NP) mode is not often used in high-performance liquid chromatography, probably because many analysts suppose that it is too complicated or even unreliable. This is by no means true rather, it is a valuable tool for the separation of a large group of analytes. Sihca, as the prototype of an NP stationary phase, is superior for the separation of isomers ds/trans, positional isomers, and diastereomers). An example is shown in Fig. 1. The silica surface is rigid (in contrast to the flexible hydrocarbon chains of the usual reversed-phase materials) and the analytes interact in a stericaUy defined maimer with the polar groups that... 

Most HPLC is based on the use of so-called normal-phase columns (useful for class separations), reverse-phase columns (useful for homolog separations), and polar columns (used in either the normal- or reverse-phase mode). Since reverse-phase HPLC columns are generally easier to work with, almost all authors use high-performance reverse-phase liquid chromatography with octade-cyl chemically bonded silica as the stationary phase and nonaqueous solvents as mobile phases (so-called NARP, or nonaqueous reverse-phase chromatography). 

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