26 August / September 2019
Figure 3. Flash Chromatography Polar Test Mix on 100-90% of B in A 3 min step gradient – Solvent A: Water with 10 mM ammonium formate; Solvent B: 95% ACN in water with 10mM ammonium formate; Teledyne ISCO 12g GOLD Silica (20-40 µm) Redisep, 30 mlmin-1, 220 nm and 254 nm.
We have invested a lot of time in developing methods for HILIC semi-prep core separations however this is a core purification service and this study has demonstrated the ability for chemists to use their own flash chromatography systems. In order to compare the silica flash column separation with that of a C18 reversed phase flash column separation, we injected the same 20mg of test mix on a RP gradient (5 to 20% ACN with 10 mM ammonium formate) as shown in Figure 4. Cytosine was not retained under these condition and purine and nicotinamide co-elute as do procainamide and tryptophan. The elution order for caffeine on the C18 flash column is totally reversed when compared to the HILIC silica flash column. This demonstrates
the utility of employing HILIC flash for these types of polar samples.
Another example of separation of an extremely polar test mix (Adenosine CLogP -1.2, Cytidine CLogP -2.2, 5’-adenylic acid CLogP -3.1) on HILIC silica (nucleoside and nucleotide) is demonstrated analytically in Figure 5, and a successful purification by flash HILIC is performed in Figure 6. This result being performed without the need for ion-pairing reagents, which is the normal approach using tetrabutylammonium hydroxide [13].
Finally, we investigated and compared different manufacturer’s analytical HILIC silica columns and alternative analytical HILIC stationary phases which are also available in flash chromatography cartridge
versions (namely Cyano, Amino and Diol). We found that Kromasil silica was virtually identical to Waters Atlantis for our test mix separation whilst we observed lower selectivity and retention on cyano and differing selectivity and retention order on amino and diol columns. This experiment demonstrates that HILIC separation may be achieved on alternative phases other than silica. The results are shown in Figure 7.
Conclusion
We have demonstrated that scaling to HILIC flash chromatography from HILIC LC-MS analytical methods is routinely achievable and can offer several advantages over generic semi-prep HPLC methods, being
Figure 4. Flash Chromatography Polar Test Mix on 5-20% of B in A for 9 min gradient – Solvent A: Water with 10mM ammonium formate; Solvent B: 95% ACN in water with 10 mM ammonium formate; Teledyne ISCO 15.5g GOLD C18 (20-40µm) Redisep, 30 mlmin-1, 220 nm and 254 nm.
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