Superheated high temperature to improve size exclusion chromatography separation of polyethylene glycols with chloroform as the mobile phase

In our laboratory, chloroform is increasingly required to be used as the mobile phase for the size exclusion chromatography (SEC) characterization of polyethylene glycol (PEG) derivatives, because some of the derivatives show poor solubility in many other solvents. Four types of SEC columns, all based on highly cross-linked polystyrene–polydivinylbenzene (PS/PDB) and compatible with chloroform, have been tried. However, a problem of using chloroform with all the columns tested is that retention might not be rationalized simply based on the SEC-mechanism even for the PEG standards. It was found that for the PEG standards raising the column temperature can significantly improve the SEC separation. In order to take full advantage of the temperature effect on separation, a system was developed which enables the SEC to be performed at superheated temperatures, i.e., temperatures well above the normal boiling point of the mobile phase. The improved SEC separation at elevated temperatures is most likely due to the combination of reduced adsorption of PEGs by the stationary phase and increased solubility of the solutes in the mobile phase. In this work, the SEC separation operated at temperatures above the normal boiling point of the mobile phase was called “superheated high temperature SEC”.

► SEC separation of PEGs can be improved significantly by raising column temperature.
► SEC at temperature above the mobile phase boiling point is called superheated HT-SEC.
► Thermal stability of an SEC column depends on the analyte chosen for the test.
► Columns with high Tmax and Pmax are needed for superheated HT SEC.
► Superheated HT-SEC can be a useful way for problematic SEC at room temperature.