Retention in RPLC

In 1996, Snyder and Dolan elaborated the linear solvent strength (LSS) approach allowing the evaluation of log few from a single gradient run and its precise determination from two gradient runs [48]. From a practical point of view, LSS is the most useful approach to describe theoretical aspects and LSS gradients are convenient for optimization studies. Several commercially available optimization software packages which are able to predict resolution or retention in RPLC are currently based on this approach. 

Proper protein folding is a concern in the production of a bioactive protein therapeutic, and it is of interest to verify that the conformation of a recombinant protein is the same as the wild-type molecule. Because retention in RPLC depends on the surface hydrophobic contact area, comparable chromatographic behavior of a recombinant protein with that of the wild-type molecule provides evidence of similar 3-D structure.17 38... 

Many drugs have either acidic or basic functional groups and can exist in solutions in ionized or non-ionized forms. The ionic state and degree of ionization greatly affect their chromatographic retention in RPLC. Typically, the ionic form does not partition well into the hydrophobic stationary phase and therefore has significantly lower k than the neutral. 

Even more than in other LC techniques, the exact mechanism of retention-in RPLC is unclear. Certainly, a simple picture that would enable us to derive unambiguous equations for the variation of retention with the various parameters of interest cannot yet be drawn. Unfortunately, there has been too much speculation in the literature throughout the last decade, often accompanied with insufficient experimental data to justify the contusions drawn. Therefore, it is not surprising that there are many different propositions for expressions to describe the retention behaviour in RPLC. 

The extent to which retention in RPLC can be made to vary with the composition of the mobile phase is enormous. For almost all solutes retention will be impractically low in some pure organic solvent (methanol, THF) and impractically high in pure water. Hence, to achieve reasonable retention times, a mixture of water and an organic solvent (a so-called modifier) is usually required. 

Unlike the relationship between retention and composition, the temperature dependence of retention in RPLC is beyond dispute. A typical van t Hoff-type equation may be used ... 

Naturally, the number of initial experiments required to start the optimization procedure will increase if either the number of parameters considered or the complexity of the model equations increases. As far as the number of parameters is concerned, we have seen this to be true with any optimization procedure, and hence the number of parameters should be carefully selected. In order to avoid a large number of initial experiments, the complexity of the model equations may be increased once more data become available during the course of the procedure. For example, retention in RPLC may be assumed to vary linearly with the mixing ratio of two iso-eluotropic binary mixtures at first. When more experimental data points become available, the model may be expanded to include quadratic terms. However, complex mathematical equations, which bear no relation to chromatographic theory (e.g. higher order polynomials [537,579]) are dangerous, because they may describe a retention surface that is much more complicated than it actually is in practice. In other words, the complexity of the model may be dictated by experimental... 

Mobile phase (eluent) is by far the major tool for the control of analyte retention in RPLC. Variations of the eluent composition, type of organic mod-iher, pH, and buffer concentration provide the chromatographer with a valuable set of variables for successful development of a separation method. 

This brief descriptive overview of the reversed-phase process emphasizes the complexity of the retention mechanism and the necessity to consider the influence of different and independent processes on the analyte retention. Since the governing process in the analyte retention is the adsorption equilibrium, the influence of the surface packing material (stationary phase) on the analyte retention in RPLC is described in Section 4.3. 

Derivatization is another form of sample preparation. It is utilized for the analysis of labile analytes or to enhance retention or detection with a preferred type of detector. Derivatization can be performed to enhance detection by UV/Vis, fluorescence, or electrochemical detection. Consideration must be given to the stability of the derivatize to solvolysis and thermal degradation. In our labs alendronate, a bisphosphonate with a primary amine functionality, was derivatized with FMOC to enhance detection by UV/Vis as well as to increase retention in RPLC mode [19]. An acylchloride was derivatized with... 

In LC-ESI-MS, the role of the mobile phase pH is complicated. In practice, often a compromise must be strack between analyte retention and ionization. From the perspective of generating preformed ions in solution, the optimum conditions for the ESI analysis of basic compounds, e.g., amines, would be an acidic mobile phase with a pH at 2 units below the dissociation constant pIQ of the analytes, while for acidic compounds, e.g., carboxylic acid or aromatic phenols, a basic mobile phase with a pH two units above the pK, of the analytes is preferred [97]. These conditions are uirfavourable for an analyte retention in RPLC. The analytes elute virtually umetained. In RPLC, it is important to reduce protolysis of basic and acidic analytes, i.e., to assure that the compounds are... 

Retention in RPLC with aqueous-organic mobile phases depends mainly on hydrophobic interactions. In general, some correlation between RPLC retention data and log is observed for a given series of solutes. The observed relationships support the assumption that in RPLC, partitionii rather than adsorption processes are decisive for retention. 

The 1-octanol-water system is challenged as the only valid hydrophobicity scale. It may not be the best model for studying the partitioning process m biomembranes. Does not the retention in RPLC model better this partitioning It has been argued that the chemical bonded stationary phase resembles more the hydrocarbon chains of membranes, but the bonding density of almost all commercial columns may be too low to provide a suitable model. 

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