For some cannabis-based product developers reversed-phase chromatography has become the analytical tool of choice for determining the extract content profile as well as for purification of specific cannabinoid compounds. However, the extracts often contain many other compounds which reduce load capacity and purity of the product(s) of interest and then require even more extensive clean-up.
In this post I show the results of an orthogonal flash purification approach that first uses normal-phase flash column chromatography to clean up the crude cannabis extract followed by reversed-phase C18 flash chromatography of the isolated target compounds. This orthogonal approach to purification increases the targeted product’s purity.
Recently, several states in the US have de-criminalized use of recreational cannabis containing the hallucinogen THC and other cannabinoids with purported medicinal value, e.g. CBD (Figure 11). However, since these compounds are naturally occurring their extracts often contain other undesirable compounds, both natural and man-made (e.g. pesticides). Current technology uses extraction with supercritical fluids or other non-supercritical solvents to remove the products of interest from other endogenous species such as lipids, terpenes, and chlorophylls as well as pesticides.
While these techniques help clean up raw extracts and isolate cannabinoids with higher-purity, the purity levels desired by processors (90%+) are not being met. So, there is now a developing need for a secondary purification step to remove the undesired co-extractants and provide the desired product purity.
To get an idea on how complex a real extract is I first tried reversed-phase flash chromatography using a 12 g C18 cartridge (Biotage® SNAP Ultra C18) and a Biotage® Isolera system. As these compounds are quite hydrophobic I chose a mobile phase gradient high in organic solvent (MeOH). The gradient started at 80% methanol in water and ended at 90% methanol.
Though very lipophilic (hexane soluble), I was able to dissolve the crude extract in DMSO at a concentration of ~0.5 g/mL. The injection volume was 0.1 mL meaning my actual load was ~50 mg of crude extract.
The chromatography is quite revealing, showing a moderately complex elution profile containing seven UV absorbing peaks, Figure 2.
Based on HPLC results reported in literature (I had no standards), I believed the major blue peak to be CBD, the major yellow peak to be THC, and the major pink peak to be THC-A1; the green peak is the DMSO solvent peak. Though this separation is good the load was quite low (50 mg) and none of the fractions were visually pure, as you can see from the chromatogram. Besides that the crude extract turned my flash cartridge brown, not ideal for future purifications.
So, in an effort to improve the reversed-phase chromatography efficiency (and keep my C18 column cleaner), normal-phase flash chromatography was employed to clean-up the extract. The thinking behind this is that potential interferences such as terpenes and colored chlorophylls found in the reversed-phase fractions can be removed by normal-phase column chromatography (many terpenes typically elute near the solvent front and chlorophylls are retained).
For normal-phase I dissolved the extract in hexanes (~0.5 g/mL) and used TLC with a hexanes/ethyl ether (9:1) solvent system2 to get an idea of what to expect in terms of the separation, Figure 3. The TLC shows streaks of UV absorbing compounds with a high concentration near Rf 0.35.
From the TLC data a normal-phase flash chromatography method was created (2-20% ether in hexanes) using a 10 g Biotage SNAP Ultra cartridge. A injection containing 250 mg of the dissolved crude was loaded (5 x more than the C18 cartridge).
The resulting normal-phase chromatography shows a good separation with one very large peak and a few smaller peaks, Figure 4. Some un-identified terpenes are believed to be found in the first two fractions with the cannabinoid acids eluting late (green fractions); no chlorophylls eluted.
Select fractions isolated from the normal-phase purification (fractions 5, 6, and 7) were each dried on a Biotage® V-10, re-dissolved in 0.2 mL of DMSO, and the entire volumes separately purified using reversed-phase flash chromatography, Figure 5.
The reversed-phase purification of normal-phase fraction 5 shows one major peak and a few minor peaks, a much cleaner profile than that in Figure 2. The MW for the major peak was determined by an Isolera™ Dalton to be 314, which is the MW for THC. The early eluting peak (blue) in figure 2 has been removed as has most of the pink peak.
Reversed-phase chromatography of normal-phase fraction 6 shows two major and a few minor peaks, also a cleaner profile than the crude. The large yellow peak also had a MW of 314 while the pink peak has a MW of 358 which matches the MW for THC-A. The blue peak has also been removed.
Normal-phase fraction 7 purification by reversed-phase shows us that there are two compounds which make up the large yellow peak in fraction 6. Both have the same MW and may be THC isomers, one being Δ9 and the other Δ8, but this has not been confirmed. Also now present is the blue peak which was found to be CBD (MW 314). This data indicates that by normal-phase, CBD is retained longer than THC, which makes sense if you look at the molecules’ structures (Figure1).
These reversed-phase results show that extract clean-up using normal-phase flash chromatography improved subsequent reversed-phase purification of the collected fractions at an increased load (~2X). It also kept my C18 cartridge cleaner and enabled more efficient purification at larger scale.
The message here is that if you are faced with purifying complex mixtures you may need to look at multiple purifications using different modes. In this case, normal-phase flash chromatography provided an excellent clean-up of a natural product extract allowing the major isolated fractions to be re-purified by reversed-phase flash at a higher load achieving better fraction purity.
1. Andrew Aubin, What’s in it & how much; Analysis of cannabinoids in plants and extracts, Waters Corp. BioBotanical Workshop, 2015.
2. N. Galand, et. al., Separation and Identification of Cannabis Components by Different Planar Chromatography Techniques (TLC, AMD, OPLC), J. Chromatographic Science, April 2004.