Gradient elution mode reversed-phase separation

Reverse phase chromatography is finding increasing use in modern LC. For example, steroids (42) and fat soluble vitamins (43) are appropriately separated by this mode. Reverse phase with a chemically bonded stationary phase is popular because mobile phase conditions can be quickly found which produce reasonable retention. (In reverse phase LC the mobile phase is typically a water-organic solvent mixture.) Rapid solvent changeover also allows easy operation in gradient elution. Many examples of reverse phase separations can be found in the literature of the various instrument companies. 

Normal-phase (NP) and reversed-phase (RP) liquid chromatography are simple divisions of the LC techniques based on the relative polarities of the mobile and stationary phases (Figure 4.10). Both NPLC and RPLC analysis make use of either the isocratic or gradient elution modes of separation (i.e. constant or variable composition of the mobile phase, respectively). Selection from these four available separation techniques depends on many variables but basically on the number and chemical structure of the compounds to be separated and on the scope of the analysis. 

This study evaluated the impurity profile of untreated water from a textile plant in Portugal [35]. The organic material was concentrated by extraction from 11 of water into dichloromethane and HPLC-NMR and HPLC-MS experiments were carried out using a reverse-phase separation with an acetonitrile/ D2O gradient elution with H NMR spectroscopic observation at 600 MHz. For the HPLC-NMR studies, the samples were further fractionated into two pools according to their HPLC retention times. The HPLC-NMR studies were carried out in the stop-flow mode and the combination of NMR and MS results yielded the identification or tentative identification of 14 compounds, comprising mainly surfactants, anthraquinone dyes and nonylphenol-related molecules. 

Separation of phthalates by EC is usually performed in the reverse phase mode using CIS columns, although the use of other stationary phases can also be found in literature. " Both isocratic and gradient elution modes were described. 

For the separation of the usual thiols (cysteine, homocysteine, cysteamine, glutathione, N-acetylcysteine), iso-cratic and gradient elution modes have been suggested using reversed phase columns [439]. 

For the optimization of nano-HPLC separations of GSL extracts, reverse-phase (RP) HPLG with a Cjg column and amidophase under different mobile phase adjustments in both isocratic and gradient elution mode can be considered for neutral and acidic GSLs. Adequate conditions for separation criteria, considering either the ceramide or the carbohydrate moiety, were elucidated using different solvent mixtures. As the matrix solvent exerts an influence on ionization efficiency, its composition must be adapted accordingly so as to achieve ionization of both the major and minor components present in the fractions. 

It is estimated that over 65% (possibly up to 90%) of all HPLC separations are carried out in the reversed-phase mode. The reasons for this include the simplicity, versatility, and scope of the reversed-phase method [23]. The hydrocarbon-like stationary phases equilibrate rapidly with changes in mobile-phase composition and are therefore eminently suitable for use with gradient elution. 

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. 

LC-ISP-MS has been also successfully applied for the assay of 21 sulfonamides in salmon flesh (121). Separation was achieved in a reversed-phase LC system with gradient elution. Simple positive-ion spectra with an intense protonated molecule and no fragment ions of relevant abundance were displayed by all analytes by operating in the full-scan acquisition and SIM modes. Further application of tandem MS using SRM for increased sensitivity could overcome the lack of structural information presented by the ISP mass spectra. 

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