Reversed-phase liquid chromatography-hydrophilic

J. Crommen and P. Herne, Indirect UV detection of hydrophilic ionized compounds in reversed-phase liquid chromatography by use of a UV-absorbing ion of the same charge, J. Pharm. Biomed. Anal., 2 241 (1984). 

Kalili, K. M. and Villiers, A. 2009. Off-line comprehensive two-dimensional hydrophilic interaction x reversed phase liquid chromatography analysis of procyanidins. J. 

In pharmaceutical development, the determination of APIs and counterions are two important assays. Due to the charge and/or hydrophobicity differences, APIs and counterions are usually analyzed by different chromatographic techniques that require different separator columns and/or detection methods. For example, reversed-phase liquid chromatography is most commonly used for analyzing APIs with intermediate to higher hydrophobicity, but it fails to provide adequate retention for hydrophilic counterions. In contrast, ion chromatography provides a selective and highly sensitive solution for the analysis of counterions. 

Since TTX is a very polar compoimd, retention on conventional reversed-phase columns is poor. Even though reversed-phase liquid chromatography is overall the most commonly used separation technique for TTX, certain TTXs are not retained. Use of an ion-pair reagent or hydrophilic interaction liquid chromatography (HILIC) can help solve this problem. Previous studies conducted with different columns and MS methods showed that 5,6,11-trideoxyTTX was not retained on a C18... 

Very frequently it is difficult to classify a separation technique as LAC or HPPLC since it is not completely clear if the sample fully precipitates in the initial mobile phase (different chemical compositions of the maaomolecules). In such cases, the separation can be classified as normal-phase liquid chromatography (NP-LC) or reversed-phase liqitid chromatography (RP-LC). In both cases, the initial and final mobile phases shoidd be good solvents for the sample. In NP-LC, a polar (hydrophilic) stationary phase is used. The initial mobile phase is less polar and the solvent strength is increased by adding a polar solvent. Typical examples are n-hexane-tetrahydrofuran (THF) and n-hexane-chloroform. RP-LC uses nonpolar (hydrophobic) stationary phases and a polar itutial mobile phase that is modified in the course of the gradient with a less polar solvent. Typical examples for this case are ACN-THF and ACN-methylene chloride. ... 

One weakness of the dominant reverse phase separations mechanism has been the poor retention of highly polar analytes, and hydrophilic interaction liquid chromatography (HILIC) has emerged as an alternative. In HILIC, a polar stationary phase such as silica gel is used to retain highly polar analytes. Mobile phases components similar to those described above for reverse phase separations are used, but the proportions of aqueous vs. organic are changed. Analytes are retained under conditions of relatively low water content, and eluted using increased water content. 

Antidepressant separation was usually performed by reversed-phase chromatography with typical C8 or C18 alkyl chain columns, although phenyl [30, 59] or cyano [48,64, 84] stationary phases were also employed. As an exception, hydrophilic interaction liquid chromatography (HILIC), a variation of normal phase chromatography, was employed in two analytical methods for duloxetine [38] and... 

Qin F, Zhao Y, Sawyer MB, Li X (2008) Column-switching reversed phase-hydrophilic interaction liquid chromatography/tandem mass spectrometry method for determination of free estrogens and their conjugates in river water. Anal Chim Acta 627 91-98... 

Micellar liquid chromatography (MLC) is another variation on reversed-phase and ion-pair chromatography. In this mode, the counter ion is a surfactant of high concentration and a long-chain hydrocarbon. Micelles form when the concentration of the surfactant is increased to the point at which aggregation occurs and spherical particles are formed. The hydrophilic parts of the long-chain hydrocarbon are oriented toward the outside of the sphere with the hydrophobic end in the center of the sphere. A mixture of compounds of varying polarity partitions between the... 

The analysis of non-polar solutes by high performance liquid chromatography is generally a simple task, especially if reversed-phase systems are used. However, many compounds of environmental interest, such as dithiocarbamate salts and inorganic anions are ionized species. By their very nature it is difficult to chromatograph these well hydrated hydrophilic species. 

Mant, C.T. and Hodges, R.S. Context-dependent effects on the hydrophilicity and hydrophobicity of side chains during reversed-phase high-performance liquid chromatography implications for prediction of peptide retention behaviour. J. Chromatogr. A. 2006, 1125,211-219. 

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