Organic acids reversed-phase chromatography

The extent to which the ions compete with B for the charged sites (X) will determine their retention. In general, this type of chromatography may be used to separate ionic species, such as organic acids or bases, which can be ionized under certain pH conditions. Besides the reaction with ionic sites on the stationary phase, retention may also be affected by the partitioning of solutes between the mobile and stationary phases, as in reversed-phase chromatography. Thus, even nonionized solutes may be retained on ion-exchange columns. 

The removal of triglycerides from the food sample by saponification provides the opportunity to utilize reversed-phase chromatography. The unsaponifiable matter is conventionally extracted into a solvent [e.g., diethyl ether/petroleum ether (50 50) or hexane] that is incompatible with a semiaqueous mobile phase. It then becomes necessary to evaporate the unsaponifiable extract to dryness and to dissolve the residue in a small volume of methanol (if methanol is the organic component of the mobile phase). For the analysis of breakfast cereals, margarine, and butter, Egberg et al. (153) avoided the time-consuming extraction of the unsaponifiable matter and the evaporation step by acidifying the unsaponifiable matter with acetic acid in acetonitrile to precipitate the soaps. An aliquot of the filtered extract could then be injected, after dilution with water, onto an ODS column eluted with a compatible mobile phase (65% acetonitrile in water). 

Offline precolumn derivatization is the most common alternative in this respect it involves separating the esters obtained from the organic acids by reversed-phase chromatography, which amply surpasses solvophobic chromatography (i.e., the use of undissociated acids as such) and allows gradient elution techniques to be applied, thanks to the wider lipophilicity range covered by the derivatized compounds. 

Based on the fact that aromatic sulfonic and carboxylic acids were successfully separated by reversed-phase chromatography in the presence of organic electrolytes, Chaytor and Heal (158) developed a method for the separation of 15 synthetic colors using a mobile phase containing o-phosphoric acid (Table 7). The presence of the electrolyte provided lower variation in response and retention over a period of time. Furthermore, eluted peaks were sharper than those seen in ion-pair chromatography. 

Nakamura, H., Kobayashi, J., and Hirata, Y., Separation of mycosporine-like amino acids in marine organisms using reverse phase high-performance liquid chromatography, J. Chromatogr., 250, 113, 1982. 

A. Tilly Melin, Y. Askemark, K.-G. Wahlund, and G. Schill, Retention behaior of carboxylic acids and their quaternary ammonium ion pairs in reversed phase chromatography with acetonitrile as organic modifier in the mobile phase, Anal. Chem. 51 (1979), 976-983. 

Reversed-phase chromatography (RPC) is a method in which molecules are bound hydrophobically to nonpolar ligands in the presence of a polar solvent. Solutes are generally bound in an acidic mobile phase with elution occurring during a gradient to an organic solvent. 

The most common mobile phases used for reversed-phase chromatography involve combinations of either methanol or acetonitrile and buffered water (like phosphate buffered, pH 7). There are other organic solvents that are used in conjunction with buffered water in reversed-phase chromatography, e.g., other alcohols or THF, but these are less commonly used. Similarly to normal phase, pH can be used to increase/decrease the interactions between the solutes and the stationary phase. Common additives are trifluoroacetic acid and diethylamine. As with normal-phase chromatography, the mobile phase must be chosen carefully. Generally, it is understood that the mobile phase should have a low viscosity, be LTV transparent, and be completely miscible. 

A significant breakthrough in cinchona-based enantioselective chromatography came with the demonstration by Lindner et al. in 1996 that immobilized quinine or quinidine 9-O-carbamates under polar organic or reversed phase condition efficiently resolved a number of acidic racemates, with opposite elution orders compared to unmodified QN or QD CSPs [44, 61]. Cinchona 9-O-carbamates contain a new functionality that can serve as a binding site of double character, that is, an H-bond donor-acceptor and, depending on the N-substituent, it may also provide a steric barrier or possibly a source of %-% interaction (Figure 13.11). 

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