Mobile phase solvating

Shifts in the SEC fractionation range are not new. It has been known for decades that adding chaotropes to mobile phases causes proteins to elute as if they were much larger molecules. Sodium dodecyl sulfate (SDS) (9) and guanidinium hydrochloride (Gd.HCl) (9-12) have been used for this purpose. It has not been clearly determined in every case if these shifts reflect effects of the chaotropes on the solutes or on the stationary phase. Proteins are denatured by chaotropes the loss of tertiary structure increases their hydrodynamic radius. However, a similar shift in elution times has been observed with SEC of peptides in 0.1% trifluoroacetic acid (TEA) (13-15) or 0.1 M formic acid (16), even if they were too small to have significant tertiary structure. Speculation as to the cause involved solvation effects that decreased the effective pore size of the... 

Mobile phases with some solvating potential, such as CO2 or ammonia, are necessary in SGC. Even though this technique is performed with ambient outlet pressure, solutes can be separated at lower temperatures than in GC because the average pressure on the column is high enough that solvation occurs. Obviously, solute retention is not constant in the column, and the local values of retention factors increase for all solutes as they near the column outlet. 

Although cSFC shows relatively poor figures of merit (speed, sensitivity, detection dynamic range and sample capacity) as well as a limited application area, its applications tend to be unique. These include solutes that can be solvated with pure SCCO2 and quantified with FID. Linear density programs typical in cSFC are ideal for homologous series found in surfactants, many prepolymers, etc. Selectivity in cSFC, which can be achieved by mobile phase density and temperature programming, relies on selective interactions with the stationary phase. Quantitative analysis in cSFC may be rendered difficult by small injected volumes the use of internal standards is recommended. 

The retention depends on the nature of both the stationary phase and the organic modifier in the mobile phase. Therefore CHI values obtained using different systems show different sensitivities towards solute characteristics. This has been studied systematically and used for the quantitative calculation of solute molecular descriptors (H-bond donor capacity, H-bond acceptor capacity and dipolarity/polarizability) for application in a general solvation equation [21]. 

The second approach was taken by practicing liquid chromatographers. They routinely dealt with thermally labile, highly polar molecules and frequently sacrificed resolution, and speed in their separations because of the aqueous mobile phases that were required. With the enhanced diffusion and decreased viscosity of supercritical fluids over liquids, chromatographic run-time and resolution could be improved when supercritical fluids were used. But solubility in pure carbon dioxide mobile phases, which has the solvating powers from hexane to methylene chloride under normal density ranges, was a problem for these polar molecules. To compensate for this, experimentalists started working with mixed mobile phases. These mixed phases were based on... 

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