centrifugal partition chromatography cbd

December 15, 2021 By admin Off

“It will definitely be an advantage to have cannabis-based medications with clearly defined and constant contents of cannabinoids,” says Kirsten Müller-Vahl, a neurologist and psychiatrist at Hannover Medical School in Germany.

‘A pharmaceutical treasure trove’

As demand for cannabis-based therapeutics increases, so will the pressure on companies and scientists to find more exact ways to isolate and purify compounds.

“Cannabis, the plant, has been described as a pharmaceutical treasure trove,” says Mark Ware, a pain medicine specialist at McGill University in Montreal, Canada. “But I think we’re at a very early stage of understanding how the individual components work in terms of their mechanism and how they interact.”

Pharmaceutical interest is focused on the plant’s many compounds, including its terpenoids, and its namesake cannabinoids — the group of related compounds that include the psychoactive agent tetrahydrocannabinol (THC) and the medicinal substance cannabidiol (CBD) — any number of which could offer treatment for or pain relief from a range of debilitating diseases.

Gilson’s CPC 250 with PLC 2250 Purification System has become a popular tool among those researching medical marijuana. Credit: Gilson.

Not everyone agrees. Many scientists and drug makers want to bring a greater rigour to the study of natural cannabinoids, and that requires the isolation of individual compounds. At the moment only chromatography allows such precision, particularly one method called centrifugal partition chromatography (CPC).

Traditionally, the stationary phase is made of silica or some other solid particle, with a liquid or gas as the mobile phase. But with CPC, the stationary phase is actually a liquid spun around to stay in one place while another liquid moves through the column.

Just like oil and water, the two liquid phases don’t mix. And with a big enough column — plus an array of liquid solvents to choose from — chemists can isolate just about any chemical component from a complex extract at purities of 99 percent or more. Gilson, the lab instruments company, sells CPCs with column sizes of 100, 250 or 1000 millilitres. The systems come packaged with software called Gilson Glider CPC and are often paired with another machine that removes residual solvent after purification.

Powers of separation.

That appeals to clinician-scientists like Kyle Geary, an internist at the University of Illinois at Chicago. He is currently running a placebo-controlled trial of CBD capsules for treating Crohn’s disease. The company behind the hemp-derived CBD product claims that levels of other cannabinoids are not detected in its analysis, but that seems unlikely for something made via CO 2 extraction.

Like all variants of column chromatography, CPC works by separating chemical substances according to their affinities for one of two ‘phases’, one stationary and one mobile. The more something sticks to the stationary phase the slower it will move through the column.

Although these molecules can be synthesized in the laboratory, many companies prefer to source the compounds from plant extracts, sometimes purifying them further by distillation. Even using this method, however, the result “is still a mixture of whatever cannabinoids are coming from a particular marijuana strain, which is a highly variable thing,” says Brian Reid, chief scientific officer of ebbu, a Colorado company that specializes in cannabis purification.

At ebbu, Reid uses a Gilson CPC 1000 system to purify various cannabinoids for research purposes and to create accurate and reliable mixtures that he sells to other cannabis product manufacturers. “Now that we have this methodology dialed in,” he says, “we can make various formulations — whether they’re water-soluble, sublingual, inhaled, you name it — with very precise ratios of cannabinoids and precise amounts of cannabinoids at the milligram level.”

“Ideally, the perfect study would use something that is 100% CBD,” says Geary, adding: “It’s hard to do studies on things you can’t control very well.”

The flexibility and robustness of CPC systems are attractive to scientists. “They just do really efficient separation of a lot of compounds at once,” says Justin Fischedick, lab director of Excelsior Analytical Laboratory, a cannabis testing company in Union City. California.

Of the hundreds of chemical compounds in cannabis, more than 100 have potential medical application. Beyond the psychoactive THC compounds, the plant’s cannabinoids are perhaps the most heavily researched. But the relatively little-known flavonoids, terpenes and fatty acids could be of use as well. Adapted with permission from Nature Research.

The extracts are not pure — but they’re not meant to be. Ethan Russo, who served for more than ten years as a senior medical advisor at GW and now directs research and development at the International Cannabis and Cannabinoids Institute in Prague, Czech Republic, says that medicinal effects result from the combination of compounds. “This poly-pharmacy inherent in a cannabis extract is a distinct advantage,” he says.

Figure 2. How CPC isolates compounds from complex, natural mixtures. As the column spins, the mobile phase (yellow) moves through each cell in series. The compounds in the mobile phase (A, B, and C) diffuse into the stationary phase (blue) at different rates according to their relative affinities for the two phases.

The mechanics of a CPC run are analogous to the mechanics of a standard elution using a traditional chromatography column. While HPLC, for instance, involves eluting cannabis oil through a resin-packed chromatography column, CPC instead elutes the oil through a series of cells embedded into a stack of rotating disks. These cells contain a liquid stationary phase composed of a commonly used fluid such as water, methanol, or heptane, which is held in place by a centrifugal force. A liquid mobile phase migrates from cell to cell as the stacked disks spin. Compounds with greater affinity to the mobile phase are not retained by the stationary phase and pass through the column faster, whereas compounds with a greater affinity to the stationary phase are retained and pass through the column slower, thereby distributing themselves in separate cells (Figure 2).

Additionally, these chromatography methods use large quantities of solvents to elute natural compounds, which negatively impacts the environment.

Most manufacturers use chromatography techniques such as high performance liquid chromatography (HPLC) or flash chromatography to isolate compounds from natural product extracts. While these methods are effective for other applications, they are not, however, ideal for CBD isolate production. Crude cannabis oil contains some 400 potentially active compounds and requires pre-treatment prior to traditional chromatography purification. Both HPLC and flash chromatography also require silica resin, an expensive consumable that must be replaced once it is contaminated due to irreversible absorption of compounds from the cannabis extract. All of these factors limit the production capacity for CBD manufacturers.

Figure 1. The structure of cannabidiol, one of 400 active compounds found in cannabis.

A Superior Chromatography Method.

Environmental Advantages of CPC.

Using a liquid stationary phase instead of silica imbues CPC with several time and cost benefits. Because natural products such as raw cannabis extract adhere to silica, traditional chromatography columns must be replaced every few weeks. On the other hand, a chemist can simply rinse out the columns in CPC and reuse them. Also, unlike silica columns, liquid solvents such as heptane used in CPC methods can be distilled with a rotary evaporator and recycled, reducing costs.

A chemist can choose a biphasic solvent system that will optimize the separation of a target compound such as CBD to extract relatively pure CBD from a cannabis extract in one step. In one small-scale study, researchers injected five grams of crude cannabis oil low in CBD content into a CPC system and obtained 205 milligrams of over 95% pure CBD in 10 minutes.

Centrifugal partition chromatography (CPC) is an alternative chromatography method that can help commercial CBD manufacturers produce greater quantities of pure CBD more quickly and cleanly, using fewer materials and generating less toxic waste. CPC is a highly scalable CBD production process that is environmentally and economically sustainable.

CBD is still considered an unsafe drug under federal law, but to meet the medical demand, 17 states in the US recently passed laws allowing individuals to consume CBD for medical purposes. A recent survey found that half of medicinal CBD users rely on the substance by itself for treatment. As doctors start using CBD to treat more patients, the demand for CBD is only expected to rise, and meeting that demand can pose challenges for manufacturers who are not used to producing such high quantities of CBD. Furthermore, as CBD-based drugs become more popular, the US Food and Drug Administration (FDA) will likely require manufacturers to demonstrate they can produce pure, high-quality products.

The solvents used in chromatography, such as methanol and acetonitrile, are toxic to both humans and the environment. Many environmentally-conscious companies have attempted to replace these toxic solvents with greener alternatives, but these may come with drawbacks. The standard, toxic solvents are so common because they are integral for optimizing purity. Replacing a solvent with an alternative could, therefore, diminish purity and yield. Consequently, a chemist may need to perform additional steps to achieve the same quality and quantity achievable with a toxic solvent. This produces more waste, offsetting the original intent of using the green solvent.

Centrifugal partition chromatography is the key to achieving high-purity, high-throughput cannabidiol production.

Many physicians today treat their patients with cannabidiol (CBD, Figure 1), a cannabinoid found in cannabis. CBD is more efficacious over traditional medications, and unlike delta-9 tetrahydrocannbinol (THC), the main psychoactive compound in cannabis, CBD has no psychoactive effects. Researchers have found CBD to be an effective treatment for conditions such as cancer pain, spasticity in multiple sclerosis, and Dravet Syndrome, a form of epilepsy.

CPC uses the same solvents as traditional chromatography, but it uses them in smaller quantities. Furthermore, as previously mentioned, these solvents can be reused. Hence, the method is effective, more environmentally-friendly, andeconomically feasible.

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As manufacturers seek to produce larger quantities of pure CBD to meet the demand of patients and physicians, they will need to integrate CPC into their purification workflows. Since CPC produces a relativelyduct on a larger scale, it is equipped to handle the high-volume needs of a large manufacturer. Additionally, because it extracts more CBD from a given volume of raw cannabis extract, and does not use costly silica or require multiple replacement columns, CPC also makes the process of industrial-scale CBD production economically sustainable. Since it also uses significantly less solvent than traditional chromatography, CPC makes it financially feasible to make the process of producing CBD more environmentally-friendly.

CPC’s Value in CBD Production.