Reversed-phase liquid chromatography -neutral

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... 

Bidlingmeyer, B.A. Separation of ionic compounds by reversed-phase liquid chromatography an update of ion-pairing techniques. J. Chmmatogr. 1980,18, 525-539. Sarzanini, C. et al.. Retention model for anionic, neutral and cationic analytes in reversed-phase ion interaction chromatography. AnaL Chem. 1996, 68,4494-4500. 

Reversed phase liquid chromatography (RPLC) allows the separation of analytes with different hydrophobicity and polarity characteristics. It has good selectivity mobile phases used in the technique contain organic solvents and small amounts of inorganic salts [149]. The effectiveness of the process depends on the hydrophobicity of the separated analytes. Charged substances must first be transformed into neutral derivatives (e.g., by adding appropriate anti-ions into the mobile phase). 

Micelles and cyclodextrins are the most common reagents used for this technique. Micellar electrokinetic capillary chromatography (MECC or MEKC) is generally used for the separation of small molecules [6], Sodium dodecyl sulfate at concentrations from 20 to 150 mM in conjunction with 20 mM borate buffer (pH 9.3) or phosphate buffer (pH 7.0) represent the most common operating conditions. The mechanism of separation is related to reversed-phase liquid chromatography, at least for neutral solutes. Organic solvents such as 5-20% methanol or acetonitrile are useful to modify selectivity when there is too much retention in the system. Alternative surfactants such as bile salts (sodium cholate), cationic surfactants (cetyltrimethy-lammonium bromide), nonionic surfactants (poly-oxyethylene-23-lauryl ether), and alkyl glucosides can be used as well. 

In the late 1970s, ionic surfactants were added for the first time to polar aqueous-organic mobile phases in reversed-phase liquid chromatography (RPLC) to form ion pairs (IPs). In IP chromatography, surfactant monomers adsorbed on a bonded silica-based stationary phase associate with neutral and ionic solutes, modifying their retention. The concentration of surfactant in the mobile phases is kept below the critical micellar concentration (CMC). In 1980, Daniel W. Armstrong reported the possibility of using... 

Monosaccharides are released by acid hydrolysis then labeled with the flurophore to allow sensitive detection 2-aminobenzoic acid (2-AA) for the neutral monosachharides, or l,2-diamino-4,5-methylenedioxybenzene. 2HC1 (DMB) for the sialic acids. Analysis by reversed-phase liquid chromatography provides reproducible quantitative results. 

This technique, in its most popular application, is a modification of reversed phase liquid-solid chromatography. It is based entirely on concentration equilibrium and can be used to separate highly polar materials with a nonpolar surface. A counter ion to the ion desired to be separated is added to the mobile phase along with a buffer to maintain ionic strength and pH. A "paired ion" is formed that is neutral and can be separated from other similar compounds by a normal reversed phase column. A diagram of how this is done is shown in Figure 19-6. 

Zhou, L., Welsh, C., Lee, C., Gong, X., Antonucci, V., Ge, Z. (2009) Development of LC chiral methods for neutral pharmaceutical related compounds using reversed phase and normal phase liquid chromatography with different types of polysaccharide stationary phases./. Pharm. Biomed. Anal., 49, 964—969. 

Vrakas, D., Giaginis, C., Tsantili-Kakoulidou, A. Different retention behavior of structurally diverse basic and neutral drugs in immobilized artificial membrane and reversed-phase high performance liquid chromatography ... 

---