27
The Effect of Column Temperature
on HILIC Retention
Column temperature is an important parameter that can also affect retention of polar analytes in HILIC [27]. The equation
that is often used is derived from chemical thermodynamics, where the equilibrium point is related to the temperature,
and is referred to as the van’t Hoff equation. In a chromatography sense the relationship between column temperature
and retention factor is often described by the following:
where:
• ΔH° = enthalpy of interaction between stationary/mobile phase and analyte
• ΔS° = entropy of interaction between stationary/mobile phase and analyte
• R = universal gas constant
• T = column temperature in Kelvin
•
= phase ratio
The van’t Hoff equation should also apply to HILIC if the retention mechanism is consistent, in which case the
relationship between ln k and 1/T is linear [27].
Using the experimental plots of ln k versus 1/T, covering a temperature range between 20–70 °C, we calculated the
enthalpic and entropic contributions to the chromatographic retention of a series of test compounds (deriving - ΔH° from
the slope and ΔS° from the intercept respectively). Temperature was observed to affect the retention and separation in
different fashions dependent on the column being tested.
On the next page are van’t Hoff plots for salicylic acid and cytosine, on four different columns. It can be seen that
a decrease in retention for salicylic acid − indicating a negative heat of enthalpy and hence an exothermic reaction
– is observed as the column temperature is increased on the two bare silica materials: Accucore HILIC and Hypersil
GOLD Silica (although on Accucore HILIC the relationship is not linear, suggesting the presence of multiple retention
mechanisms). On the zwitterionic phase−Syncronis HILIC−the retention of salicylic acid is more or less independent
of temperature; this behavior suggests low enthalpic contribution and high entropic contribution to the retention.
Interestingly, the retention increases with the temperature on Hypersil GOLD HILIC. Positive enthalpy was obtained for this
anion exchanger, indicating an endothermic process of transferring salicylic acid from the mobile phase to the stationary
phase. For Hypersil GOLD HILIC, the positive enthalpy of salicylic acid could also be the evidence of both ion exchange
and partitioning processes taking place.
ln k= - H°/RT + S°/R + ln
ln k= - H°/RT + S°/R + ln
