Development and application of a specially designed heating system for temperature-programmed high-performance liquid chromatography using subcritical water as the mobile phase

Abstract

A specially designed heating system for temperature-programmed HPLC was developed based on experimental measurements of eluent temperature inside a stainless steel capillary using a very thin thermocouple. The heating system can be operated at temperatures up to 225 °C and consists of a preheating, a column heating and a cooling unit. Fast cycle times after a temperature gradient can be realized by an internal silicone oil bath which cools down the preheating and column heating unit. Long-term thermal stability of a polybutadiene-coated zirconium dioxide column has been evaluated using a tubular oven in which the column was placed. The packing material was stable after 50 h of operation at 185 °C. A mixture containing four steroids was separated at ambient conditions using a mobile phase of 25% acetonitrile:75% deionized water and a mobile phase of pure deionized water at 185 °C using the specially designed heating system and the PBD column. Analysis time could be drastically reduced from 17 min at ambient conditions and a flow rate of 1 mL/min to only 1.2 min at 185 °C and a flow rate of 5 mL/min. At these extreme conditions, no thermal mismatch was observed and peaks were not distorted, thus underlining the performance of the developed heating system. Temperature programming was performed by separating cytostatic and antibiotic drugs with a temperature gradient using only water as the mobile phase. In contrast to an isocratic elution of this mixture at room temperature, overall analysis time could be reduced two-fold from 20 to 10 min.