The Role of Temperature in HPLC

We have seen in the previous sections that retention in chromatography follows the rules of equilibrium (or reversible) thermodynamics. This is the foundation for the very important effect of temperature on retention and sometimes also 

1 Retention and Selectivity Control via Temperature Possibilities and Limitations 

The general influence of temperature on chromatographic retention can be explained as follows. In order to be retained on the stationary phase, molecules need to transform from a state of very little order while floating in the mobile phase to a much more ordered immobilized state in the stationary phase. This is a process that implies a reduction in entropy. In order to make retention still an energetically favorable process, thermodynamics defines that it must release heat. Thus, retention in chromatography is typically an exothermic process. If the temperature is increased, exothermic processes escape from this constraint by shifting the equilibrium to the original side. This implies that temperature increase is accompanied by a shift to the desorbed state of the molecules and thus a lower retention. There are some rare exemptions to this rule (as always), but those are based on secondary equilibria that overrule the effect described earlier. 

Retention in chromatography is controlled by thermodynamic equilibria. The partition ofthe analyte between the mobile and the stationary phase is in control of the retention factor. This partition can be described by the laws of reversible thermodynamics. Therefore, we also borrow the thermodynamic description of the temperature dependence of equilibria. This is the so-called van t Hoff equation, which is the quantitative expression of the Le Chatelier principle. According to this, the temperature dependence of the retention factor k can be described by 2.9, with R being the general gas constant, AH° the molar enthalpy (heat tone) related to the transition of the analyte from mobile to stationary phase, AS° the molar entropy change for this transition, andj( the so-called phase ratio of the packed stationary phase in the column. 

The following rules of thumb apply for retention and selectivity optimization  

---