How to Control Selectivity in HPLC

To address this essential question, the triangle of interactions in hquid chromatography has to be initially investigated. While the mobile phase in GC has only the function to transport the analyte through the column without undergoing any interactions, the situation in LC is far more complex. The interactions between analyte molecules and the stationary phase (same as in GC) have to be considered, as well as the interactions between the mobile phase molecules and the stationary phase. In a simple model, we can assume that mobile phase molecules and analyte molecules compete for spaces on the stationary phase. This competition is expressed in thermodynamic equilibria and thus also controlled by the temperature. Last but not least, there is also the interaction between analyte molecules and mobile phase molecules to be taken into consideration. 

Experienced HPLC users with a reasonable chemical knowledge will have individual strategies for selectivity improvement. These are commonly based on assessment of the relative changes of molecular interactions between analytes and the surface of the stationary phase, as well as analytes and mobile phase components. All possible physicochemical interactions such as London dispersion forces, dipole interaction, hydrogen bonds, coulomb interaction,. r-electron interactions, or complex formation are present in LC. The order given relates 

It is also important to understand, that retention in RP-LC is never solely based on a pure hydrophobic or dispersion interaction, but always influenced by secondary retention mechanisms. These secondary mechanisms can be both of wanted or of an unwanted nature. A good understanding of these relationships is extremely helpful for selectivity optimization and the fundamentals are discussed in more detail in Chapter 4 on modern HPLC columns. 

Before we discuss the influence of pH, additives, and temperature in more detail, we need to understand another important factor for optimization of peak resolution. Peak resolution is affected by the difference in retention time between peaks and the peak shape. We have discussed the intrinsic effect of band dispersion in chromatography, which leads to a symmetrical broadening of peaks. This peak width is described in a standardized way by the plate number. In practical LC, however, perfectly symmetric peaks are rather the exception, and it is an important criterion of method optimization to remove root causes for peak distortion. Every increase in peak asymmetry negatively affects resolution (under otherwise constant conditions). It must be emphasized in this context that Eq. 2.1 in Section 2.2.1 is only valid for Gaussian peaks with perfect symmetry, while any peak distortion will lead to smaller effective resolution. There are cases where the 

Selectivity Control by Mobile Phase pH and Column Temperature These two parameters are of major importance for selectivity control in HPLC, especially with acidic and/or basic analytes. The charge state of analyte molecules influences both their hydrophobic (dispersion type) interaction, as well as possible ionic secondary interactions (e.g., with dissociated residual silanol group on silica-based stationary phases). The relatively wide range of so-called mixed-mode phases make very effective use of ionic interactions, but specialty phases are outside the scope of this chapter (refer to Chapter 4 for more information). 

---