Method development for the separation of sulfonamides by supercritical fluid chromatography

Supercritical fluid chromatographic separation of eight regulated sulfonamides is achieved within 20 min by coupling a packed-silica column (250 × 4.6 mm) to an aminopropyl-packed column (250 × 4.6 mm) employing ultraviolet detection. The chromatographic conditions are as follows: 10% methanol is used for 5 min, then increased 2.5% per minute to 30% methanol. The column oven temperature is 65°C, the outlet CO2 pressure is 300 bar, and the liquid flow rate is 2.5 mL/min. Other stationary phases investigated are Amino 2, Deltabond CN, and Alltima CN (all columns are 250 × 4.6 mm, 5-μm dp). Column deactivation is found to decrease the resolution of the polar sulfonamides by decreasing analyte interaction with active sites. The retention time of sulfathiazole is found to decrease by nearly 50% using an Amino 2 deactivated column, compared with a Spherisorb amino column. Retention behavior of some of the analytes is greatly affected by a subtle change in temperature.