Impact of CO2-solvent separators on the degradation of benzyl-2,3-dihydroxypiperidine-1-carboxylate during preparative supercritical fluid chromatographic (SFC) purification.

During a preparative separation of the cis enantiomeric pair of benzyl-2,3-dihydroxypiperidine-1-carboxylate using supercritical-fluid chromatography (SFC) with methanol modifier, significant degradation of the products in the collected fractions was observed when a Waters SFC-350® (Milford, MA, USA) was used, but same was not observed when a Waters SFC-80q® (Milford, MA, USA) was used. Through a systematic investigation, we discovered that the compound degraded over time under an acidic condition created by the formation of methyl carbonic acid from methanol and CO2. The extent of the product degradation was dependent on the time and the concentration of CO2 remained in the product fraction, which was governed by the efficiency of CO2-methanol separation during the fraction collection. Hence, we demonstrated that the different designs of CO2-solvent separator (high pressurized cyclone in Waters SFC-350® and low-pressurized vortexing separator in Waters SFC-80q®®) had a significant impact on the degradation of an acid-sensitive compound. The acidity caused by CO2 in methanol was supported by diminished degradation after a nitrogen purging or after neutralizing the collected fractions with a base. Three different solutions to overcome the degradation problem of the acid sensitive compounds using SFC-350® with the high pressurized separator were investigated and demonstrated. The degraded products were isolated as four enantiomers and their relative stereochemistry were established based on 2D NMR data along with the plausible mechanism of degradation.

Integrating a post-column makeup pump into preparative supercritical fluid chromatography systems to address stability and recovery issues during purifications.

Purification of many pharmaceutical compounds by supercritical fluid chromatography (SFC) has always been challenging because of degradation of compound during the isolation step in the presence of acidic or basic modifiers in the mobile phase. Stability of such acid or base-sensitive compounds could be improved by post-column addition of a solvent containing base or acid modifier as counter ion through a make-up pump respectively to neutralize the compound fraction without affecting the resolution. One such case study has been presented in this work where the stability of a base-sensitive compound was addressed by the addition of acidic co-solvent through the make-up pump. Details of this setup and the investigation of degradation of the in-house base-sensitive compound are discussed in this paper. In addition, poor retentivity and low recovery of many non-polar compounds in SFC eluting under low co-solvent percentage is another major concern. Even though the desired separation could be achieved with low percentage of co-solvent, it's difficult to get the proper recovery after purification due to precipitation of the sample and significant aerosol formation inside the cyclone. We have demonstrated the first-time use of a post-column make-up pump on SFC 350 system to introduce additional solvent prior to cyclone to avoid the precipitation, reduce the aerosol formation and thus improve the recovery of non-polar compounds eluting under less than 10% of co-solvent.