Why Centrifugal Partition Chromatography is a Better Way to Extract Cannabidiol for Medical Research

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Cannabidiol (CBD), a cannabinoid, shares some of the medicinal benefits of tetrahydrocannbinol (THC), but it does not cause euphoria or intoxication, making it an attractive subject for medical research and treatment. In fact, researchers have discovered that CBD is useful in three therapeutic areas: cancer pain, spasticity (chronic muscle contractions), and childhood seizures.^1-3 A drug composed of CBD (Figure 1) and THC (Sativex, GW Pharmaceuticals, Carlsbad, CA) has been approved in Europe and Asia to manage cancer pain and spasticity. Beyond these indications, CBD shows great promise for addressing a wide range of conditions, including neurodegenerative diseases, substance abuse, and chronic pain.²

Figure 1 – Chemical structure of cannabidiol.

As patients’ and physicians’ interest in cannabis products increases, so does the need for quality control. The FDA does not currently regulate CBD production, and the only farm in the U.S. licensed to produce cannabis is not subject to external quality control.⁴ Scientists need access to pure, research-grade CBD that is free of THC and other contaminants that could diminish the safety and reliability of their work. Thus, it is important to optimize the extraction of CBD from its source to ensure maximal recovery of pure compound.

The Cannabis sativa plant from which CBD is derived contains over 500 different compounds.⁵ Traditional chromatographic methods, such as HPLC and flash chromatography, are not suited for the needs of CBD researchers. Use of either method on a continuous basis to purify CBD from such a complex, natural product is time-consuming, cost prohibitive, and not scalable. The silica resin used in these purification systems is a costly consumable that must be replaced every few weeks or months because it sticks easily to natural products. Also, each technique requires a minimum of two steps to arrive at purified CBD, which slows the process.⁶

Centrifugal partition chromatography

An alternative method for purifying CBD and other cannabis extracts for research use is centrifugal partition chromatography (CPC). It is less expensive than conventional chromatography methods since it uses a liquid stationary phase instead of silica. Consequently, CPC columns can be reused after a simple rinse, and there is no danger of residues that can reduce the quality of the extract. The technique also saves time by requiring only one 30-minute step after the crude cannabis extract is injected into a CPC column.

Use of CPC for CBD extraction

Like HPLC, CPC uses a stationary phase and a mobile phase. While both methods share the same underlying biochemical principles, they differ in the nature of the stationary phase. Traditional chromatography uses silica as the stationary phase, and compounds in the liquid phase are eluted according to their affinity to the silica. CPC, on the other hand, uses one of several liquids commonly found in the laboratory—such as water, methanol, or heptane—as the stationary phase. The liquid stationary phase is held in place by centrifugal force, and the compounds in the mobile phase are eluted in order of their partition coefficient between the two phases.

The partition coefficient of a compound in a CPC setup is determined by observing how it distributes itself between the mobile and stationary phases.⁷ While the centrifuge is in motion, the mobile phase is pushed through the stationary phase in a series of specially designed cells that keep the latter phase in place. As the mobile phase passes through each cell, solutes are left behind in the stationary liquid.

The higher the affinity a compound has for the stationary phase, the earlier it will bind to it; subsequently, compounds with different affinities for the stationary phase will separate into different cells as the mobile phase moves through each one (Figure 2).⁷ In a system containing a water mobile phase and a butanol stationary phase, for example, if the solute is more polar, most of it will remain in the water; if the solute is more nonpolar, it will mostly diffuse into the butanol.

Figure 2 – CPC process. Mobile phase (yellow) is pushed through the stationary phase (blue) across a series of cells. The solutes (A, B, and C) in the mobile phase are left behind in separate cells according to their respective affinities to the stationary phase.

For CPC, the ideal partition coefficient, calculated as the concentration of the solute in the stationary phase divided by the concentration of the solute in the mobile phase at equilibrium, is between 0.5 and 5. In this range, the solute of interest will diffuse into the stationary phase and extract itself from the crude oil. By varying the composition of the two phases, the partition coefficients of the solutes can be fine-tuned to ensure the selective extraction of the target compound. CPC can be used to purify not only CBD, but other cannabis components, such as THC, tetrahydrocannabinolic acid, and cannabidiolic acid, all of which are highly soluble in nonpolar liquids, such as alkanes.

Besides operating by the same biochemical principles, CPC also uses the same pumps, injectors, inline detectors, fraction collectors, and other peripheral equipment found in traditional HPLC. However, because the liquid phase in CPC occupies less volume than silica, more crude extract can be injected in each run, and less solvent is needed to elute compounds contained within it. In fact, CPC uses 3–5 times less solvent than traditional chromatography methods.⁸ The solvent can be recycled, which further reduces the cost of the purification.

In addition to being less costly and easier to use, CPC also reliably yields a purer compound. Scientists extracted 205 mg of 99% pure CBD from 5 g of crude cannabis extracts using the CPC 250 system (Figure 3, Gilson, Inc., Middleton, WI). Purity was determined using analytical HPLC at 275 nm.⁹ This study involved just a single step: Crude extract of C. sativa was directly injected into the column and centrifuged.

Figure 3 – CPC 250 with PLC 2250 purification system. An analytical HPLC platform is used to analyze the CPC extracts.

Typically, CPC extracts products with nearly 100% purity, meeting the standards of traditional methods. But its recovery rate of more than 90% is higher than the average recovery rate for HPLC.⁹ In addition to CBD, Gilson has shown similar success extracting other compounds from natural sources, such as [6]-gingerol from ginger.¹⁰

CPC in the laboratory

While CPC has proven ideal for extracting CBD from cannabis, it has a few limitations. For instance, the technology is unknown to most purification specialists, and researchers may not fully understand how to optimize CPC protocols for their own laboratories. Also, CPC may not be suitable for every situation: Because it is not an analytical technique, an analytical HPLC platform is necessary to produce quality chromatograms for identifying CBD.

Gilson Purification (Vannes, France) houses an applications laboratory that instructs users on how to incorporate CPC into their CBD purification workflow. For more information, visit http://www.gilson.com/en/AI/Products/80.320.

References

1. https://www.fda.gov/downloads/aboutfda/ centersoffices/officeofmedicalproductsandtobacco/cder/ucm498077.pdf
2. https://www.drugabuse.gov/about-nida/legislative-activities/testimony-to-congress/2016/biology-potential-therapeutic-effects-cannabidiol
3. http://medicalmarijuana.procon.org/view.resource.php?resourceID=006473
4. http://www.pbs.org/newshour/updates/scientists-say-governments-pot-farm-moldy-samples-no-guidelines/
5. http://pubs.acs.org/doi/abs/10.1021/acs.jnatprod.5b00949
6. http://www.biotage.com/news/effective-cannabinoid-purification-by-flash-chromatography
7. http://www.gilson.com/Resources/Gilson_Introduction_CPC.PDF
8. http://www.sciencedirect.com/science/article/pii/S187439001730071X
9. https://cdn.technologynetworks.com/tn/Resources/pdf/cpc-250-purification-of-cannabidiol-from-cannabis-sativa.pdf
10. http://www.gilson.com/Resources/CPC_250_Purification_of_Gingerol_TN207.pdf

Gregoire Audo is director of Portfolio Management, CPC Business Line, Gilson Purification SAS, 22 rue Bourseu, ZA du Poteau, F-56890 Saint-Avé, France; tel.: +33 (0) 2 97 61 84 00; e-mail:   [email protected]; www.gilson.com