Exploring the application limits of different hold-up time markers in supercritical fluid chromatography

The study focuses on the application range of nitrous oxide as a hold-up time marker in supercritical fluid chromatography (SFC). This compound has been suggested a decade ago to be used as unretained marker, something that the field of SFC was missing for a long time, since its beneficial properties make it an ideal candidate as hold-up time marker. Determination of the hold-up volume and actual volumetric flow rates have always been problematic in SFC due to the compressibility of carbon dioxide and one part of this is the difficulty of hold-up time measurements. Depending on the mobile phase, different methods have been used to measure the hold-up time with varying results. Nitrous oxide and other molecules have been compared in different conditions, mobile phases and stationary phases. In all cases, nitrous oxide gave the lowest elution times. However, detection was difficult in mobile phases containing 10% or more of organic modifier, because most solvents mask the signal of nitrous oxide. Interestingly, the choice of stationary phase also had a slight effect on detection, while different pressure and temperature settings affected each compound in a different manner.