Mass-Directed Preparative SFC for the Isolation of a 0.2% Impurity

Download PDF August 25, 2017

Introduction

Supercritical fluid chromatography (SFC) is a rapidly growing technique. Initally used almost exclusively only for thermally labile and chiral compounds, it is now accepted as a versatile analytical and purification technique applied across a broad range of chiral and achiral compounds. It offers higher-throughput than regular HPLC and Prep-LC and offers the ‘green’ advantages of much lower solvent consumption, consumable and disposal costs. SFC is compatible with a wide range of detection methods including UV, electrospray (ESI) and atmospheric pressure chemical ionization (APCI) mass spectrometry.

Objective

To determine the purity of collection of a small (0.2%) impurity in a concentrated sample matrix using extracted ion chromatogram (XIC) for mass directed fraction collection.

Methods

  1. Pre-Purification Analytical: A small aliquot is injected to determine whether the compound target mass is present and to measure the UV purity of the sample prior to purification. The combination of target mass being found and the sample’s purity determine whether a sample moves to the preparative stage and preparative narrow method, based on target retention time, will be automatically selected for the purification process.
  2. Purification:  20 injections of 0.7 mL were made and the target mass extracted using fraction collection based on the method identified in Pre-purification. Mass directed triggering using XIC for Flavone m/z 223.1.
  3. Post Purification Analytical: A small aliquot is injected from each tube of collected fraction material and the final sample purity measured.

Experimental

Mass spectrometer:expression CMS   
SFC:Jasco Prep SFC System   
Total Flow:70 ml/min   
BPR:120 bar   
Column:Waters Viridis Silica 2-Ethylpyridine OBD Prep Column, 100A, 5 μm, 19 mm x 250 mm   
Co-solvent:Methanol   
Gradient:5-25% in 7 min.   
Splitter Configuration: 100cm (2 x 50 cm) PEEKSil -0.025 mm ID x 1/16' OD coupled with ZDV union   
Makeup pump flow:8 mL/min Me0H (constant flow for 30% modifier and below)   
Interface:Jasco ChromNav set uo to read two analog outputs from expression CMS mapped to the total ion chromatogram (TIC) nad one selected extracted ion chromatogram (XIC)mg/mL   
Sample:Flavone 9.56 mg/mL Carbamazepine 9.56 mg/mL Sulfamethazine 9.48 mg/mL   

Instrument Set up

Results

0.2% Flavone in 10mg/mL Carbamazepine

Zoom Chromatogram of 0.2% Flavone in 10mg/mL Carbamazepine.

MS Triggering with XIC

Conclusion

  • The CMS single quad MS can be simply interfaced to both analytical scale and prep scale SFC using a simple passive split before the UV detector.
  • The fractions collected for each analyte were tested analytically and showed that each was 99.9% pure or greater.
  • Limit determination of 0.2% Flavone in the presence of Carbamazepine – Using an XIC signal to trigger the fraction collector, a 0.2 wt % peak of Flavone was collected at 100% purity.

About the Author

DJ Tognarelli received his B.S. from the University of Richmond where he studied Analytical Chemistry. He is a Chromatography Applications Scientist at JASCO.