3
Figure 3. Addition of 0.1% n,n-diethylmethylamine to MeOH demonstrates superior peak shape and reversal of elution order.
There is no uniformly accepted ‘generic’ SFC column chemistry within the industry and therefore there is a need to select a range of chemistries in comparison to RP-HPLC where C18 is the more standard and unifi ed approach. A demonstration of this is shown in Figure 4 for a test mix separation for 6 different column chemistries using the same generic SFC gradient.
Figure 5. 42mg injection on 100 x 30mm Reprosil-NH2 100A 3um using 19-27% MeOH shallow gradient at 150ml mL min-1
.
Figure 4: Testmix Selectivity on range of SFC column chemistries. Strategy for Purifi cation
Based on the 2 minute gradient analytical result, we then can develop a ‘bespoke’ shallow gradient window for individual samples defi ning the percentage of MeOH needed to try and elute the compound of interest in the middle of the prep gradient. We have optimised the purifi cation run times by the implementation of higher fl ow rates and also routine use of 3um SFC Prep columns to maximise peak effi ciency. An example is shown in Figure 5 where the purifi cation total run time is only 4 minutes using a 10-27% MeOH shallow gradient at total fl ow150ml mL min-1
and has been enabled by the recent upgrade of two waters SFC-100-MS instruments by ABsys (Oberursel, Germany).
These ABsys upgrades have enabled the laboratory to increase fl ow rates of up to 180 mL min-1
, optimise gas-liquid separation to enhance recovery, shorten preparative methods, shorten equilibration times and implement waste collection for every run. The system is shown in Figure 6. This has enabled Achiral SFC purifi cation processes to be optimised and has enabled an increase in the scale of separations we can employ and has been adopted by our lab as our purifi cation system of choice.
Figure 6. ABsys upgrade to Waters SFC-100 purifi cation - GLS = Gas Liquid Separator BRP = Back Pressure Regulator
Conclusions . This is now standard procedure within our laboratory
By taking advantage of the advances in SFC technology, chromatographers are also able to capitalise on the improvements improving achiral separation effi ciency whilst also reducing environmental impact. Purifi cation via SFC is ~3 to 4x faster than a traditional RP purifi cation with purity equivalent to RP-HPLC. With the introduction of the ABsys SFC systems the reliability, speed and scale of SFC Achiral purifi cations has been improved. We in Novartis are now able to effi ciently separate achiral novel drugs whilst signifi cantly decreasing run times, environmental impact, maximising recovery with reduced dry down times. Analytical and Preparative Achiral SFC is standard practice and adopted by the whole Global Discovery Chemistry community. RP-HPLC will still remain a complimentary orthogonal technique for purifi cation and fi rst method of choice in Open Access purifi cation.
References
1. Kaljurand M., Koel M. “Recent Advancements on Greening Analytical Separation” Critical Reviews in Analytical Chemistry 2011, 41: 2-20, 2. Goetzinger W., Zhang X., Bi G., Towle M., Cherrak D and Kyrano J.N “High throughput HPLC/MS purifi cation in support of drug discovery” International Journal of Mass Spectrometry 2004, Vol 238, Issue 2, : 153-162 3. Blom, K.F, Glass B., Sparks R. and Combs A.P. Preparative LC-MS Purifi cation: Improved Compound –Specifi c Method Optimization” 2004, Journal of Comb. Chem. 6, 874-883 4. Francotte E. SFC China 2015 (Green Chemistry Group Presentation) SFC: A Multipurpose Approach to Support Drug Discovery 5. Desfontaine V., Guillarme D., Francotte E., Novakova L. “Supercritical fl uid chromatography in pharmaceutical analysis” Journal of Pharmaceutical and Biomedical Analysis,113 2015 56-71
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