Using automatic peak hold to separate partially co-eluting compounds

Summary

A novel feature that inserts an isocratic hold into a linear gradient when a compound elutes can improve the separation of partially co-eluting compounds, simplifying flash chromatography method development and optimization.

Background

Complex reaction mixtures and natural product extracts are sometimes challenging to purify due to co-eluting or partially resolved compounds. An advanced feature of the Biotage® Selekt flash chromatography system is its ability to automatically apply an isocratic hold when a new compound elutes.

By automatically inserting an isocratic hold during peak elution, the gradient slope is altered and delayed, allowing later-eluting compounds to interact with the media longer. This increased residence time can improve the separation of otherwise hard-to-separate compounds.

Results and discussion

To illustrate the power of the automatic isocratic hold capability, a 12-gram Biotage® Sfär C18 column was used with a 70-100% linear gradient of water and methanol to separate a hemp extract. Hemp extracts typically contain a broad variety of cannabinoids, which can be challenging to separate and purify.

The 10-column volume (CV) 70-100% methanol gradient successfully separated cannabidiol (CBD) from most of the other cannabinoids, but was unable to separate two other major cannabinoids, Figure 1. Rather than spending time optimizing the method through trial and error or literature references, the extract was purified using the same linear gradient but with Peak Hold activated, Figure 2.

Figure 1. Linear gradient purification failed to fully separate the major cannabinoids with several co-eluting (circle).

Figure 2. Adding the automatic Peak Hold to the linear gradient.

Peak Hold lowered the gradient slope giving the extract’s compounds more time to stick to the C18 media. This increased residence time helped to separate closely eluting and even co-eluting compounds, Figure 3. While this modified linear gradient separated several close eluting/co-eluting cannabinoids, it also increased both time and solvent consumption (31 CV vs. 16 CV). This was resolved by further optimizing the method using the Biotage® Selekt system’s Gradient Optimization tool.

Figure 3. Hemp extract purification using a 70-100% methanol linear gradient with automatic Peak Hold activated increased the separation of the later eluting compounds.

Gradient Optimization converts a previous purification into a step gradient targeting a single compound. In this application, the compound eluting between 16 and 19 CV was targeted. The resulting gradient, an isocratic method of 82% methanol, completely separated all the compounds in only 15 CV, Figure 4.

Figure 4. Gradient optimized isocratic method provided a full separation of the extract’s cannabinoids in only 15 CV.

Conclusion

Purification methods using automated Peak Hold feature can help separate partially co-eluting compounds, while further Gradient optimization can reduce both purification time and solvent use.

Literature Number: AN1014