Impact of modified carbon dioxide mobile phases on detection on packed-column supercritical fluid chromatography

TR Number



Journal Title

Journal ISSN

Volume Title


Virginia Tech


The advantages of supercritical fluid chromatography (SFC) and supercritical fluid extraction (SFE) which utilize supercritical fluids (SF) as the mobile phase are being realized by the scientific community. SF are more advantageous than traditionally used organic solvents because SF exhibits the solvating strength of liquids while maintaining the higher mass transport properties of gases. Currently, the most common SF which is CO₂ is available in two preparations of pure CO₂ and helium headspace CO₂. SFC of polyaromatic hydrocarbons (PAH) was performed with pure carbon dioxide and helium headspace carbon dioxide at various cylinder fill levels. The capacity factors of the PAH's increased when helium headspace carbon dioxide was used as a carrier fluid relative to pure carbon dioxide. As more liquid carbon dioxide was removed from the cylinder, the effect of helium on the solvating power of CO₂ was reduced because the relative amount of helium dissolved in the liquid phase decreased. Furthermore, the effect of helium headspace carbon dioxide was investigated with methanol-modified carbon dioxide mobile phases for the analysis of steroids. The capacity factors of the steroids increased when helium headspace CO₂ was used relative to pure CO₂.

Although two types of carbon dioxide can be utilized, both have found widespread use in packed-column SFC. Unfortunately, the activity of the stationary phase of packed-columns may prevent the elution of moderately polar analytes when using 100% carbon dioxide. The stationary phase activity can usually be overcome by adding a modifier or modifier with an additive. Unfortunately, the introduction of modifier/additive can interfere with the use of most commonly used detectors like the flame ionization detector (FID) and ultraviolet detector (UV). Therefore, a detector is needed in which the modifier/additive does not interfere and detection is possible. Two such detectors are the electron capture detector (ECD) and evaporative light scattering detector (ELSD).

Response surfaces were obtained for packed-column SFC-ECD under various detector conditions to optimize the ECD for use with modified CO₂. Limits of detection, correlation coefficient, and linear dynamic range were found to vary with increasing amounts of modifier for several nitrogen containing and halogenated compounds. Low detection (pg) was achieved in the presence of 5% methanol-modified CO₂. Applications of packed column SFC-ECD to the separation of nitrogen containing compounds extracted from propellants, phenylurea herbicides, and felodipine extracted from a sustained release tablet by SFE are presented.

A high performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD) has been modified and interfaced with SFC. The detector performance was evaluated by monitoring the response of several steroids. Specifically, the effect of N, make-up gas flow rate, CO₂ modifier type, modifier concentration, ELSD orifice size, and detector temperature was determined. As the N₂ gas flow rate was increased the response of the analyte decreased, but the increased flow improved the peak shape to mimic that seen by ultraviolet detection. Furthermore, increasing detector temperature caused the response of the analytes to decrease. A detection limit of 10 ng or less was determined for progesterone and testosterone using 2% and 20% (v/v) methanol-modified CO₂ on a Deltabond® cyano column (4.6 mm X 15 cm, 5 um) at 150 mL/min and 1000 mL/min decompressed CO₂. Using the detector's optimized conditions, the separations of polyethylene glycols and ginkgo biloba leak extract are reported.