Tag Archives: phase

A More Effective and Efficient Approach to Purer Cannabidiol Production Using Centrifugal Partition Chromatography

By Lauren Pahnke
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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.

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.

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

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.

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

A Superior Chromatography Method

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.

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).

Figure 2- CPC
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.

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.

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.

Environmental Advantages of CPC

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.

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.

CPC’s Value in CBD Production

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.

Suggested Reading:

CPC 250: Purification of Cannabidiol from Cannabis sativa

Introduction to Centrifugal Partition Chromatography

Terpene_KAS2
From The Lab

The Other Side of Cannabis: Terpenes

By Dr. Zacariah Hildenbrand, Allegra Leghissa, Dr. Kevin A. Schug
1 Comment
Terpene_KAS2

Have you ever wondered why all beers have that strong, characteristic smell? Or why you could tell the smell of cannabis apart from any other plant? The answer is simple – terpenes.

These 55,000 different molecules are responsible for a majority of the odors and fragrances around us, from a pine forest, to the air diffuser in your house 1–3. They all share the same precursor, isoprene, and because of that, they are all related and have similar molecular structures. Unfortunately, it is this uncanny similarity that makes their analysis so challenging; we still lack a complete list of which terpenes expected to be found in each given plant species 1,2.

Many different methods have been developed in an effort to provide a time-optimized and straightforward analysis. Gas chromatography (GC) is usually center stage due to the volatility of the terpenes. Therefore, there is significant concern with the type of GC detector used 2.

The flame ionization detector (FID) is a good quantitative detector for GC, but qualitatively it does not provide any information, except for retention time; the differentiation between terpene species is achieved solely by use of retention indices (RI), which are based on elution times from a particular GC stationary phase. The best part of the FID is its low cost, reliability, and relatively easy interface, which make it an effective tool for quality control (QC) but less so with respect to research and discovery 2.

The primary choice for a research setting is the mass spectrometer (MS) detector. It is more expensive and complicated than FID, but importantly, it provides both good quantitative capabilities, and it provides mass spectra for each species that elutes from the chromatograph. However, for terpene analysis, it may still not be the best detector choice. Since terpene class molecules share many structural and functional similarities, even their fragmentation and sub-sequential identification by MS may lead to inconsistent results, which need to be confirmed by use of RI. Still, MS is a better qualitative analysis tool than the FID, especially for distinguishing non-isobaric terpenes 2.

Recently, new technology based on vacuum ultraviolet spectroscopy (VUV) has been developed as a new GC detector. The VUV detector enables analysis of virtually all molecules; virtually all chemical compounds absorb light in the range in the 125-240 nm wavelength range probed by the detector, making it an essentially universal detector 4–11. Previously, spectroscopic absorption detectors for GC have lacked sufficient energy to measure absorption of most GC-amenable species. The VUV detector fills a niche, which is complementary to MS detection in terms of the qualitative information it provides.

Terpene_KAS2
Figure 1: A, Section of the chromatographic separation of a terpenes standard mix; B, highlight of the co-eluting terpenes, camphor and (-)-isopulegol; C, differences in the absorbance spectra of camphor and (-)-isopulegol.

With the VUV detector, each compound exhibits its own unique absorbance spectrum. Even isomers and isobars, which are prevalent in terpene mixtures and can be difficult to distinguish different species by their electron ionization mass spectra, can be well differentiated based on their VUV spectra 6,9,10.  Nevertheless, because analytes exhibit different spectra, it is not required to achieve a perfect chromatographic separation of the mixture components. Co-eluting peaks can be separated post-run through the use of library spectra and software inherent to the instrument 4,10. This ability is called “deconvolution”, and it is based on the fact that two co-eluting terpenes will give a peak with an absorbance spectrum equal to the sum of the two single absorbance spectra 4. Figure 1 shows the deconvolution process for two co-eluting terpenes, camphor and (-)-isopulegol. Due to their different absorbance spectra (Figure 1C), it is possible to fully separate the two peaks in post-run, obtaining sharp peaks for both analytes 6.

The deconvolution process has been shown to yield precise and accurate results. Thus, chromatographic resolution can be sacrificed in favor of spectroscopic resolution; this enables the development of methods with faster run times. With the ability to deconvolve unresolved peaks, a long temperature ramp to chromatographically separate all isomeric terpenes is not required 6. Additionally, the presence of coeluting components, which might normally go undetected with some GC detectors, can be easily judged based on comparison of the measured spectra with pure reference spectra contained in the VUV spectral library.

The other issue in terpenes analysis is the extraction process. Terpenes can be extracted with the use of solvents (e.g., methanol, ethanol, hexane, and cyclohexane, among others), but the process is usually time-consuming, costly and not so environmentally-friendly 2. The plant needs to be manually crushed and then aliquots of solvent are used to extract components from the plant, ideally at least 3 times and combined to achieve acceptable results. The problem is that some terpenes may respond better to a certain solvent, making their extraction easier and more optimized than for others 2. The choice of solvent can cause discrimination against the extraction some terpenes, which limits the comprehensiveness of analysis.

Headspace is another technique that can be used for the sample preparation of terpenes. Headspace sampling is based on heating the solid or liquid sample inside a sealed vial, and then analyzing the air above it after sufficient equilibration. In this way, only volatile analytes are extracted from the solid/liquid sample into the gas phase; this allows relatively interference-free sampling 12–14.

How do we know whether our extraction analysis methods are correct and comprehensive for a certain plant sample? Unfortunately, there is not a complete list of available molecules for each plant species, and even if two specimens may smell really similar to our nose, their terpenes profiles may be notably different. When working with a new plant material, it is difficult to predict the extraction efficiency for the vast array of terpenes that may be present. We can only perform it with different extraction and detection methods, and compare the results.

The route for a comprehensive and fast analysis of terpenes is therefore still long; however, their intoxicating aromas and inherent medicinal value has provided a growing impetus for researchers around the world. Considering the evolving importance of Cannabis and the growing body of evidence on the synergistic effects between terpenes and cannabinoids, it is likely that newly improved extraction and analysis methods will be developed, paving the way for a more complete list of terpene species that can be found in different cultivars. The use of new analytical technologies, such as the VUV detector for GC, should aid considerably in this endeavor.


References:

[1]          Breitmaier E., Terpenes: Flavors, Fragrances, Pharmaca, Pheromones. John Wiley & Sons 2006.

[2]          Leghissa A., Hildenbrand Z. L., Schug K. A., A Review of Methods for the Chemical Characterization of Cannabis Natural Products. J. Sep. Sci.2018, 41, 398–415 .

[3]          Benvenuto E., Misra B. B., Stehle F., Andre C. M., Hausman J.-F., Guerriero G., Cannabis sativa: The Plant of the Thousand and One Molecules. Front. Plant Sci2016, 719, DOI: 10.3389/fpls.2016.00019.

[4]          Schug K. A., Sawicki I., Carlton D. D., Fan H.,Mcnair H. M.,Nimmo J. P., Kroll P.,Smuts J.,Walsh P., Harrison D., Vacuum Ultraviolet Detector for Gas Chromatography. Anal. Chem.2014, 86, 8329–8335 .

[5]          Fan H.,Smuts J., Walsh P.,Harrison D., Schug K. A., Gas chromatography-vacuum ultraviolet spectroscopy for multiclass pesticide identification. J. Chromatogr. A2015, DOI: 10.1016/j.chroma.2015.02.035.

[6]          Qiu C.,Smuts J., Schug K. A., Analysis of terpenes and turpentines using gas chromatography with vacuum ultraviolet detection. J. Sep. Sci.2017, 40, 869–877 .

[7]          Leghissa A., Smuts J., Qiu C., Hildenbrand Z. L., Schug K. A., Detection of cannabinoids and cannabinoid metabolites using gas chromatography-vacuum ultraviolet spectroscopy. Sep. Sci. Plus2018, 1.

[8]          Bai L.,Smuts J., Walsh P., Fan H., Hildenbrand Z., Wong D., Wetz D., Schug K. A., Permanent gas analysis using gas chromatography with vacuum ultraviolet detection. J. Chromatogr. A2015,1388, 244–250 .

[9]          Skultety L., Frycak P., Qiu C.,Smuts J., Shear-Laude L., Lemr K., Mao J. X., Kroll P., Schug K. A., Szewczak A., Vaught C., Lurie I., Havlicek V., Resolution of isomeric new designer stimulants using gas chromatography – Vacuum ultraviolet spectroscopy and theoretical computations. Anal. Chim. Acta2017, 971, 55–67 .

[10]       Bai L., Smuts J., Walsh P., Qiu C., McNair H. M., Schug K. ., Pseudo-absolute quantitative analysis using gas chromatography–vacuum ultraviolet spectroscopy–a tutorial. Anal. Chim. Acta2017, 953, 10–22 .

[11]       Schenk J., Nagy G., Pohl N. L. B., Leghissa A., Smuts J., Schug K. A., Identification and deconvolution of carbohydrates with gas chromatography-vacuum ultraviolet spectroscopy. J. Chromatogr. A2017, 1513, 210–221 .

[12]       Van Opstaele F., De Causmaecker B., Aerts G., De Cooman L., Characterization of novel varietal floral hop aromas by headspace solid phase microextraction and gas chromatography-mass spectrometry/olfactometry. J. Agric. Food Chem.2012, 60, 12270−12281 .

[13]       Hamm S., Bleton J., Connan J., Tchapla A., A chemical investigation by headspace SPME and GC-MS of volatile and semi-volatile terpenes in various olibanum samples. Phytochemistry2005,66, 1499–1514 .

[14]       Aberl A., Coelhan M., Determination of volatile compounds in different hop Varieties by headspace-trap GC/MS-in comparison with conventional hop essential oil analysis. J. Agric. Food Chem.2012, 60, 2785−2792 .