The Application of SepaFlash Strong Anion Exchange Chromatography Columns in the Purification of Acidic Compounds

Results and Discussion

Firstly, the sample mixture was separated by a normal phase flash cartridge pre-packed with regular silica. As show in Figure 3, the two components in the sample were eluted from the cartridge one after another. Next, a SAX flash cartridge was utilized for the purification of the sample. As show in Figure 4, the acidic Component B was completely retained on the SAX cartridge. The neutral Component A was gradually eluted from the cartridge with the elution of the mobile phase.

Figure 3. The flash chromatogram of the sample on a regular normal phase cartridge.

Figure 4. The flash chromatogram of the sample on a SAX cartridge.
Comparing Figure 3 and Figure 4, the Component A has inconsistent peak shape on the two different flash cartridges. To confirm whether the elution peak is corresponding to the component, we can utilize the full wavelength scanning feature which is built into the control software of SepaBean™ machine. Open the experimental data of the two separations, drag to indicator line on the time axis (CV) in the chromatogram to the highest point and the second highest point of the elution peak corresponding to the Component A, and the full wavelength spectrum of these two points will be automatically shown below the chromatogram (as shown in Figure 5 and Figure 6). Comparing the full wavelength spectrum data of these two separations, the Component A has consistent absorption spectrum in two experiments. For the reason of the Component A has inconsistent peak shape on two different flash cartridges, it is speculated that there is specific impurity in the Component A which has different retention on the normal phase cartridge and the SAX cartridge. Therefore, the eluting sequence is different for the Component A and the impurity on these two flash cartridges, resulting inconsistent peak shape­­­­­­­­ on the chromatograms.

Figure 5. The full wavelength spectrum of the Component A and the impurity separated by normal phase cartridge.

Figure 6. The full wavelength spectrum of the Component A and the impurity separated by SAX cartridge.

If the target product to be collected is the neutral Component A, the purification task can easily be completed by directly using the SAX cartridge for elution after sample loading. On the other hand, if the target product to be collected is the acidic Component B, the capture-release manner could be adopted with only a slight adjustment in the experimental steps: when the sample was loaded on the SAX cartridge and the neutral Component A was completely eluted out with normal phase organic solvents, switch the mobile phase to methanol solution containing 5% acetic acid. The acetate ions in the mobile phase will compete with the Component B for binding to the quaternary amine ion groups on the stationary phase of SAX cartridge, thereby eluting the Component B from the cartridge to obtain the target product. The chromatogram of the sample separated in ion exchange mode was shown in Figure 7.

Figure 7. The flash chromatogram of the Component B eluted in ion exchange mode on a SAX cartridge.

In conclusion, acidic or neutral sample could be rapidly purified by SAX cartridge combined with normal phase cartridge utilizing different purification strategies. Furthermore, with the help of full wavelength scanning feature built into the control software of SepaBean™ machine, the characteristic absorption spectrum of the eluted fractions could be easily compared and confirmed, helping researchers quickly determine the composition and purity of the eluted fractions and thus improving work efficiency.