Chromatography gradient formation

High-pressure liquid chromatography (HPLC) formats that use reverse-phase analytical columns dominate the bioanalytical field. Isocratic elution formats are used for traditional LC-MS/MS bioanalysis, since these separations do not require additional time for column reequilibration. However, gradient elution is a more effective approach for cassette analysis in highthrough-put screening methods for discovery applications because of its versatility for the separation of compounds that have a wide variety of lipophilicity. 

Adsorption chromatography using small particle silica or alumina has also been employed in the separation of biologically meaningful substances. Phospholipids, for example, have been separated on silica (38). One of the big problems for such substances is detection, since many of the compounds are not U.V. active. Generally, the refractive index detector is employed for isocratic operation, and the moving wire detector for gradient operation. Formation of U.V.-active derivatives is also possible (39). 

The popularity of reversed-phase liquid chromatography (RPC) is easily explained by its unmatched simplicity, versatility and scope [15,22,50,52,71,149,288-290]. Neutral and ionic solutes can be separated simultaneously and the rapid equilibration of the stationary phase with changes in mobile phase composition allows gradient elution techniques to be used routinely. Secondary chemical equilibria, such as ion suppression, ion-pair formation, metal complexatlon, and micelle formation are easily exploited in RPC to optimize separation selectivity and to augment changes availaple from varying the mobile phase solvent composition. Retention in RPC, at least in the accepted ideal sense, occurs by non-specific hydrophobic interactions of the solute with the... 

Figure 26-7 Anion separation by ion chromatography with a gradient of electrolytically generated KOH and conductivity detection after suppression. Column Dionex lonPac AS11 diameter = 4 mm flow = 2.0 mL/min. Eluent 0.5 mM KOH for 2.5 min, 0.5 to 5.0 mM KOH from 2.5 to 6 min 5.0 to 38.2 mM KOH from 6 to 18 min. Peaks (1) quinate, (2) F, (3) acetate, (4) propanoate, (5) formate,...

However CCR reduction in SCT SRC occurs with significant alteration of GEC classes, primarily toward the formation of less polar compounds. The classes of W. Kentucky SCT SRC, separated by GEC (gradient elution chromatography), are given in Table 2 and show that 75% of the SRC is polar and noneluted polar asphaltenes. 

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