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Figure 1. HILIC Analytical Polar Test Mix on 100-60% of B in A for 6 min gradient – Solvent A: water with 10 mM ammonium formate; Solvent B: 95% ACN in water with 10mM ammonium formate; Atlantis HILIC Silica column 4.6x100mm, 5µm.
HPLC-MS as a “Scout” for HILIC Flash Purification
A similar approach was undertaken to the reversed-phase methodology previously published [12] where we demonstrated the efficiency of predicting flash chromatography purification conditions from an analytical HPLC scout run. For HILIC we now selected a polar test mix using small molecule polar compounds, nicotinamide, nucleic bases purine and cytosine, amino acid tryptophan and small basic molecules caffeine and procainamide.
We decided to use this polar test mix separation to determine whether we can scale up to HILIC silica flash chromatography. We utilised a 12g Teledyne
ISCO Gold silica cartridge (20-40 µm) and observed the separation in Figure 2 for a 20 mg total loading. We realised we needed to extend the gradient time out to 9 minutes from the 6 minutes employed on the analytical HPLC column to achieve baseline separation on the flash column.
This result was extremely encouraging as this demonstrates we can enable chemists to separate extremely polar analytes on flash instead of utilising semi-prep HPLC. Technology advancements have led to flash purification being a relatively cost effective solution where inexpensive plastic cartridge columns are used to often purify larger batches of material quickly in one purification run. A further advantage is that the capital and running costs for a standard
flash chromatography system make this an attractive financial solution for many laboratories for scaled up separations, and reduced fraction volume in comparison to HPLC due to sample loading increase is a real benefit for dry-down time of samples [12]. This type of HILIC gradient flash separation has yet to be utilised as a method for purification in medicinal chemistry laboratories, but is advantageous due to better theoretical sample loading than reversed phase HPLC. The Gold silica flash columns are re-useable and similar separation is achieved even after 10 injections. Using a stepwise gradient approach can also be beneficial to achieve further retention and provide a separation profile as seen in Figure 3.
Figure 2. Flash Chromatography Polar Test Mix on 100-60% of B in A for 9 min gradient – Solvent A: water with 10 mM ammonium formate; Solvent B: 95% ACN in water with 10 mM ammonium formate; Teledyne ISCO 12g GOLD Silica (20-40µm) Redisep, 30 mlmin-1, 220 nm and 254 nm.
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