Reversed Phase Liquid Chromatography RP-LC

Reversed-phase liquid chromatography (RP-LC) has been proposed as an alternative method for log determination, simulating the partition of a specific compound between the lipid layer and the biological membrane of a cell. This is the process which rules the biological activity of substances, such as the absorption of drugs and their metabolites, the bioaccumulation of organic compounds, and so forth. 

Reversed-phase liquid chromatography (RP-LC) separates analytes based on the differences in their interactions with the non-polar stationary phase. In this mode, the stationary phase is less polar than the mobile phase. The reversed-phase stationary phases often contain alkyl groups (C2, Cg, or Cjg). 

The selectivity of reversed-phase liquid chromatography (RP-LC) columns is known to vary, even columns with the same ligand (e.g., Cjg). Column selectivity can also vary from batch to batch for columns claimed to be equivalent by the manufacturer. For different reasons, it is sometimes necessary to locate a replacement column for a given assay that will provide the same separation as the previous column. In other cases, as in HPLC method development, a column of very different selectivity may be needed - in order to separate peaks that overlap on the original column. For each of these situations, means for measuring and comparing column selectivity are required. Until recently, no such characterization of column selectivity was able to guarantee that two different columns can provide equivalent separation for any sample or separation conditions. 

GC, gas chromatography NP-LC, normal-phase liquid chromatography RP-LC, reversed-phase liquid chromatography SFC, supercritical fluid chromatography IC, ion chromatography CE, capillary electrophoresis SEC, size exclusion chromatography. 

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. ... 

Overall, it is well understood that one method cannot cover the whole metabolome, so the current state-of-the-art demonstrates the need for the application of a number of analytical techniques such as reversed-phase liquid chromatography-mass spectrometry ((RP)LC-MS) untargeted as a general profiling tool, another RPLC-MS method to profile the lipids (on the C30 or C18 column), GC-MS to profile the content of volatiles, and one or two LC-MS or CE-MS methods (targeted and untargeted) to profile the polar metabolites (see Section 5.1). 

Liquid chromatography coupled to electrospray ionization mass spectrometry (LC-ESI-MS) was introduced in the 1980s [1]. Today it has become a standard method for separation and characterization of nonvolatile compounds. Reversed-phase high-performance liquid chromatography (RP-HPLC) coupled to ESI-MS is the method of choice for peptide and protein analysis, but also used for the characterization of contaminants, therapeutic drugs, and food additives [2-5], More than 75% of HPLC analyses are run on RP stationary phases, and a wide range of columns are available with various substituents of the silica matrix, base deactivation, endcapping, and column dimensions. 

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. 

LC-NMR. Separations using reverse-phase (RP) liquid chromatography are potentially more powerful because samples can be studied without derivatization. Numerous attempts have been made to separate NOM and while most studies exhibit some degree of separation, to date the complete separation of a NOM sample has not been accomplished. Even only partial separation is possible, and it is worth to hyphenate a separation method with structure information-oriented analytical applications. Liquid chromatography combined with nuclear magnetic resonance and preliminary studies with solid-phase extraction were conducted on NOM isolated from freshwater and soil (Simpson et al., 2004). 

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