Impact of column temperature on triacylglycerol regioisomers separation in silver ion liquid chromatography using heptane-based mobile phases

This work presents the investigation of the use of heptane as an alternative and less toxic mobile phase to the most used hexane for triacylglycerols (TAGs) analysis in silver ion high-performance liquid chromatography (Ag+-HPLC). The impact of column temperature (in the 5 °C-35 °C range) on the retention and resolution of five pairs of regioisomers relevant for the confectionery industry was investigated using a heptane-based mobile phase modified with acetonitrile (ACN). The retention behaviour was compared for a standard TAG mixture and an interesterified cocoa butter. The temperature effect previously observed with hexane-based mobile phases was confirmed for this new system, and it was also observed that the ACN concentration had an important impact on the strength of the temperature effect, with a higher ACN concentration leading to a lesser impact of temperature on the TAGs' elution behaviour. In general, the study allowed to conclude on the equivalence of hexane and heptane for TAGs regioisomers separation in Ag+-HPLC, independently of the used temperature or the ACN concentration. In addition, the applicability of heptane-based mobile phases for the separation of TAGs regioisomers was demonstrated on three other confectionary fat samples, namely palm olein, interesterified palm olein, and interesterified shea olein.