Purification methods: Cannabinoid Recrystallization

Growing Crystals.

One pretty reliable recrystallization technique involves a mixed solvent system. For this, you will need two solvents of different strength: one that dissolves the molecules easily (Ethyl Acetate) and another that the molecules are mostly insoluble in (Pentane).

Experiment at a small scale.

A good working sample size is around 20-200 mg, and a 20 mL dram vial with a screwcap, or a small round bottom flask with a stopper are ideal. To the evaporated solid sample in the vial, a few mL of pentane are added and then broken up with a stirring rod or metal spatula, ideally sonicated, to produce a fine suspension of the solid. Then, with stirring and/or sonicating, Ethyl Acetate was added one drop at a time until the cloudiness dissipates to a clear, transparent solution. Let the vial sit at room temperature in an undisturbed ventilated area overnight, loosely capped, allowing a slow evaporation to occur. If crystals form in the next few days, they can be collected and washed with cold pentane.

The Layering Method.

If the evaporation leaves behind more of a residue, you can try a layering method. Re-dissolve the sample in the same way that you did before, but before you cap it, using a pipette, add a few mL of pentane slowly and carefully along the side of the vial to make a distinct layer on top. The crystals will form at this layer. Close the cap tightly this time, and let it sit undisturbed in a freezer for a few days. You can try several different ratios of solvent or temperatures. Once you figure out a method that works, you can try that on a larger scale. If your sample is not pure enough for recrystallization, other isolation methods, such a chromatography, may be necessary beforehand.

Contact Us.

If you’re interested in learning more about recrystallization as a method for purifying your extracts, CannaChemist can help. Contact us at info@oriongmp.com and let us help you.

This content is written and supported by Orion GMP Solutions, a pharmaceutical engineering firm dedicated to international standardization of GMP Cannabis.

Validating SOPs for GMP Cannabis

The objective of validating a procedure is to demonstrate that the procedure is suitable for its intended purpose. This extends to all SOPs. They must be validated to prove that they accomplish their purpose. There are many different processes that can be validated in pharmaceutical operations. Some examples include, but are not limited to, process chemistry, analytical testing, lab facilities, cleaning, equipment, packaging, etc.

For the sake simplicity, this article will cover validation of analytical methods. Method development and validation are all about setting specifications and making sure that the method can reliably achieve those standards. The specifications are discovered during method development, where an analyst works by trial and error to find the right conditions, that are described by example below. It is a tedious process, but once the proper method for analysis is established (i.e. the right column, the right flow rates, the right wavelength, and right temperatures), you have data that should show a reproducible method. From there, it’s a matter of setting the amount of variance that is tolerable to still accomplish the method (i.e. validation parameters).

Analytical method development is the time when the robustness of a method is established. Robust in this sense, means that you can change parameters of the method without seeing variation in the results – that is, despite conditions being less than optimal, you still get good results. Validation checks the variation in methods – you must get the same results for a given method within a specified percentage or relative standard deviation. If a method has been proven to be robust, it has a much greater chance of passing validation (being within the specified variance).

There are three major types of analytical methods: identity tests, assays, and impurity tests. An identity test proves that a certain molecule is present in a sample. An assay shows how much of a molecule is present in a sample. An impurity test shows how much of the sample has degraded or the relative quantities of impurities present in a sample. There are 6 major parameters tested in the validation of analytical methods: accuracy, precision, specificity, detection limits, quantification limits, and range.

Validation parameters require qualified reference standards. Ideally they will be from a third party, manufactured in an ISO environment that ensures the purity. The qualified reference standards are how meaningful comparisons are made to assess each parameter.

accuracy – how close to the target value the method reliably achieves
precision – how close each measurement is to the other measurements in a series of measurements
specificity – identification of the exact molecule that’s being tested – i.e. the method can discriminate between molecules similar to the target molecule.
detection limit – the smallest quantity of a molecule that can be detected
quantification limit – the smallest quantity of a molecule that can be reliably quantified
range – the smallest and largest amount of a molecule that can be reliably quantified in an analytical test

Details of the method should be clearly listed and explained in the validation report. They are important because they clearly lay out the conditions to execute a given method. Here are a few examples of method conditions:

Description of the method – e.g. HPLC
Type of chromatography column – e.g. C18 Reverse Phase HPLC Column
Flow rate and method durations – e.g. 1mL/min – 20 min runtime
Detection Wavelength – e.g. 210nm
Column Temperature – e.g. 30C

If you have more questions, check out www.oriongmp.com and get a free consultation on putting together your Cannabis related Good Manufacturing Practices and Quality Manufacturing Systems.

GMP Training for Cannabis Manufacturing and Testing

GMP training is an important part of manufacturing and testing pharmaceutical products. It is the means of ensuring employees can properly perform their job to specified standards. Those standards ensure products are manufactured consistently, in a controlled manner, and to defined quality specifications. Training employees on a regular schedule, with job specific instruction, and thorough documentation help companies meet their standards of manufacturing and testing of their pharmaceutical products.

The first component to a quality training program is that training occurs on a recurring basis. When a new employee joins a company, it is necessary for them to learn, understand, and be tested on their job specific duties. After initial training they are qualified to do their job. They will then be required to perform refresher training that makes sure they are still up to date with any changes to the processes used for their job. For example, if a process is changed, the employee needs to have documented training on the change, proving they understand the change and can employ the process properly.

The second component to a quality training program is that the training covers the scope of an employee’s job. Each employee has their own specialized tasks in a manufacturing or testing facility, and they are responsible for knowing their job as it’s defined by the company. For example, a quality control chemist does not need to know the intricacies of a process chemist’s job, and vice versa. Each respective chemist needs to fully understand their own job so they can perform it up to the standards set in training.

Recurring training is a standard element of any quality process in the pharmaceutical industry. The proof of that training is in documentation. Documentation consumes a large amount of time in a quality manufacturing and testing environment. It is not fun and few people like it. It can take more time than the work itself. Still, without documentation, there is no proof of what has been done. It is important that companies have a documented training program that outlines each job in the company. Each job will have specific duties associated with it, and each duty requires training.

This article isn’t meant to touch on the qualifications of individuals bring to a job, but it grazes an interesting point. People who may not be qualified to start in a job can become qualified. An employee may not know how to operate a Gas Chromatography (GC) instrument when they start the job, but if they know how to operate an HPLC, there’s little reason why they can’t be trained to run a GC. On the job training (OJT) is a viable way to build up your employees’ capabilities and careers. When OJT is well documented, it’s the proof of a person’s education. It’s not only important to prove that your process is being executed by properly trained employees, but it’s also important to your employees’ careers.

If you have more questions, check out www.oriongmp.com and get a free consultation on putting together your Cannabis related Good Manufacturing Practices and Quality Manufacturing Systems.

10 Commandments for Cannabis Testing Facilities

GMP Cannabis, THC, and CBD are on their way to the market. This is a fact. In order for labs to stay on top of the market, they must adapt to the practices of the pharmaceutical industry. This is a basic list of 10 things cannabis testing labs should already be doing.

Thou shalt not perform analysis without proper training and understanding of the equipment. Qualifying a properly trained technician is dependent on the job they are doing. Have they been trained on the instrument? Have they taken sufficient analytical chemistry courses to understand how to interpret results? An analyst must know how to interpret the results, or figure out why the results they are get are inconsistent. If the results do not make sense, the analyst must troubleshoot the instrument based on what the data is telling them and then find out what went wrong. This type of critical and analytical thinking requires a thorough training program for all analysts.
Thou shalt not perform laboratory analysis without Standard Operating Procedures (SOPs) that are followed exactly as they are written. This assumes that a laboratory has done a good job of writing their SOPs in the first place. SOPs define exactly how a test will be performed; this reduces any variation in process that could affect the results. SOPs also define the technique that an analyst should use – for example, are all analysts in the lab using the same mass or volume measurement techniques? If there is variance in the process, there will be variance in the results.
Thou shalt not perform laboratory analysis using SOPs that have not been validated. In order to reduce variations in results, a method (e.g. the way you analyze your sample by HPLC) must produce the same results reliably. A validation is a set of experiments that is used to prove that the method does in fact produce the same results that are within the specifications of the desired method.
Thou shalt not perform laboratory analysis without accurately weighing and measuring the samples. This also falls under the category of training, but is perhaps the most important factor in accurately and reliably analyzing samples. This is also the most difficult technique to master, because there are so many variables. The best way to accurately weigh and measure samples is to perform any weighing and measuring the same way every time it is done. Exactly the same way – no excuses. Everyone in the lab should also weigh and measure samples with the exact same technique. This reduces the amount of variability in results throughout the lab.
Thou shalt not perform laboratory analysis without accurately documenting the results from weighing and measuring. This is self explanatory, but it may not be done this way in every lab. I have had the misfortune of seeing an analyst simply weigh out a sample, pour in ethanol up to an arbitrary line, shake it up, and inject the sample into an HPLC. He never recorded the weight of the sample or the volume of liquid that was added, and didn’t account for either of them… This kind of sloppiness is what makes labs untrustworthy.
Thou shalt only use analytical reference standards (ARSs) that have been produced by an ISO accredited lab. Creating in-house reference standards is possible, but it is highly unlikely that your lab has the capabilities of qualifying them. An accredited ISO lab synthesizes THC/CBD/etc and has exact ways of measuring the quantities that are in their ARSs. If you have variability, or uncertainty, in the amounts of THC/CBD/etc in your ARSs, your analysis will not be accurate. Reliable ARSs are the backbone of good analysis.
Thou shalt inject your ARS’s at a minimum of 5 times for any number of samples. This is how to determine if your instrument is working within its specifications/calibration, and includes the chromatography column and the instrument in general. If there is a poor Relative Standard Deviation (RSD), i.e. the 5 injections of the ARS are not within a specified percentage (think precision), the analysis of the samples is disregarded.
Thou shalt calculate the Relative Standard Deviation of the 5 ARS injections. This is done by taking the average of the response factors for a given peak (e.g. THC), dividing it by the standard deviation, and multiplying by 100%. For a very tight group of data, an analyst with good preparation technique and a well calibrated instrument will pull 2% RSD. RSD is the means of showing just how precise your techniques and instruments are running, and is a very important tool for tracking the performance of your work.
Thou shalt establish correction factors for different cannabinoids being analyzed. Not all cannabinoids will have the same response factor in the same chromatography instrument. This is particularly true for spectrophotometry (e.g. HPLC), that uses light to detect the presence of molecule. Since different molecules will absorb different amounts of light, the apparent amounts/concentrations will be thrown off if they are not corrected for.
Thou shalt document everything. All analysis must be documented with the weight or measures of the samples that are used, and contain all data collected from each test. The records must contain the calculations used to draw conclusions, and must make a statement of the results. The test results must be compared to qualified reference standards that clearly show identity, strength/potency, quality, and purity of the drug product.

If you have any comments or questions, please post them in the comments section or email andrew@hemphacker.com.

Pure THC and CBD – Green Chemistry

This question was asked in the comments section in cannabis extracts in Reddit – how do you produce pure Δ9 THC that’s appearing in dispensaries??? A fellow named Adam Mueller answered this question, and figured out the sub/supercritical conditions to produce pure Δ9 THC and was issued a patent for it last year.

It is also possible to extract and purify THC to a high concentration using other methods. However, they mostly use solvents that are known to be carcinogenic. CO2, on the other hand, defines a “Green Solvent” – no carcinogenicity, and no waste products that pollute the environment.

Patent: US 2014/0248379 – Process for producing an extract containing THC and CBD from cannabis plant material, and cannabis extracts.

Adam Mueller has several patents describing the extraction of cannabinoids from hemp and drug varieties of cannabis. He represents Delta-9-Pharma GmbH, a company out of Germany. While this information may not be novel to people who are already performing extracts, it is of interest to people who would like to learn about the conditions.

This patent explains the steps from taken from grinding up plant matter, subjecting it to subcritical or supercritical conditions and producing a pure CBD and THC product. An interesting point is that CBD can be converted to THC with the right conditions. Past that, the product is dewaxed and ready for delivery in the activated decarboxylated form.

There are some aspects of the patent that are very useful, but there are downsides to running supercritical CO2, given what we know about the “entourage effect” described by Ethan Russo (1). Cannabinoids alone do not have the highest medicinal benefits as a mixture terpenes and cannabinoids. While this patent describes a way to obtain highly purified CBD and THC, the terpenes end up in a separate fraction. Recombining THC, CBD, and terpenes is not unheard of, but it does require a bit more work.

Raw materials

Mueller’s primary source of raw materials is hemp. This is a logical starting material for countries where it is illegal to cultivate marijuana. For the validation of the patent, he used five different strains that are from French, Hungarian, and Finnish origins. In general, these are industrial hemp varieties that are used for fiber production. The legal requirements for such strains are that they contain no more than 0.3% THC by dried weight of the starting materials.

Sub/Supercritical conditions

Mueller provides a range of conditions that can be used to extract cannabinoids with supercritical CO2. Supercritical conditions range from 31-80℃ and 75-500 bar. Subcritical conditions range from 20-30℃ and 100-350 bar.

Subcritical and supercritical extracts do not come out with the same consistencies. Subcritical extractions preserve more terpenes, while supercritical extractions lose terpenes and increase the waxes. Subcritical extracts have an oil like consistency, have less plant lipids (waxes), and may not need dewaxing, depending on the application and what customers want – it is nearly ready for use in vape pens or to be dabbed. Supercritical extracts have a more solid “crumble” consistency, have more plant lipids, and most certainly require dewaxing in order to be used in vape pens or dabs. Both can be used for edibles.

A follow up point is that both subcritical and supercritical runs can be done on the same starting material. First perform a subcritical run and collect the extract – the extract will still have some terpenes present without being overwhelmed with waxes. Then, the same starting material can be run a second time under supercritical conditions. Running subcritical conditions produces relatively low yields, so to maximize yield (i.e. profitability) one really needs to run supercritical conditions. You may find the extra work of doing two runs is not worthwhile, considering you have to have to do twice the work. There’s always a trade-off somewhere in a process, and this one is for you to decide. For maximum yield, Mueller suggests performing two runs on the same material.

Mueller increases yields by using “entraining agents.” Butane, propane, and ethanol are used in concentrations of 1-10%.

Entraining agents have different properties than supercritical CO2. The critical point (CP) of CO2 is 73.8 bar and 31.5℃; butane has a CP of 38.0 bar and 152℃ (2); propane’s CP is 42.5 bar and 96.7C (3); ethanol’s CP is 63 bar and 241℃. A mathematical description requires computational chemistry to show how the entraining agents interact with the CO2. Suffice it to say, CO2 will be in the supercritical phase and the entraining agents will be in the liquid phase (3), (4). This changes the solvent characteristics of the CO2 and improves extraction yields.

His optimal supercritical conditions range from 45-65℃, 100-350 bar, with his best conditions being 60C and 250 bar. His preferred subcritical conditions range from 20-30℃ and 100-350 bar.

Mueller illustrates the system in figures 1-3. It’s a complicated system that is appropriately named a “CO2 extraction plant.” This system was designed for process scale. The system has three components: the extraction system (figure 1), the CBD cyclization system (figure 2), and the CBD/THC separation system (figure 3).

How CO2 supercritical work – solubility

The extraction starts in the extraction vessel (F1: 1-4). The column(s) is packed with plant material, and the sub/supercritical CO2 begins to strip the plant material of cannabinoids. The cannabinoids are called the solute, and the CO2 is called the solvent. A solvent becomes saturated when it has no more “room” for more solutes to be carried by the solvent.

The cannabinoid saturated solvent then passes into the separating vessels (F1: 5a-5b) in a continuous process. Fresh solvent enters the extraction vessel, dissolves cannabinoids and other phytochemicals, and carries them into the separating vessels. The cannabinoids are the first solutes to pass through the separating vessels, followed by terpenes, and then the undesirable phytochemicals.

The order of the solutes entering the separating vessel (packed with adsorbents) is dependent on how strong of an interaction each molecule has with the adsorbent media. Cannabinoids have the weakest interaction (first out) and the undesirable phytochemicals have the strongest (last out).

The solubility of the different solutes in the solvent depends on both pressure (P) and temperature (T). The general scheme of the system is to go from high P to low P, and high T to low T, in graduated steps. At high P and T have the highest solubility.

As the P and T are reduced, the solubility of certain components is reduced. For example, the extraction vessels and separating vessels run at 60℃ and 250 bar. The terpene/cannabinoid rich solvent is then pumped over to the first collection vessel (10), at 45℃ and 60-75 bar. That reduction in P and T causes the terpenes to fall out of solution. Meanwhile, the cannabinoids are still in solution.

The cannabinoids are then pumped to the next collection vessel (14). T and P are reduced to 20℃ and 50 bar. Under these conditions, the cannabinoids fall out of solution, and are ready for collection.

Dewaxing and decarboxylation

Dewaxing is pretty standard in the industry these days. Unless you run very cold conditions, you’re going to pick up wax in both hydrocarbon and CO2 extractions. To put it simply, cold ethanol is used to dissolve the extract, followed by freezing and filtration. The extract can then be moved on to decarboxylation, which is a well documented process in the industry.

Converting CBD to THC

The second admirable part of this patent is the cyclization reaction of CBD to THC. Molecular/zeolytic sieves and zinc chloride are employed as a catalyst to aid the reaction. The molecular sieves act as a water binding agent, and the zinc chloride acts as a catalyst to reduce the activation energy required for the cyclization reaction.

The conditions are 300 bar and 70℃, and the reaction takes place over a 2 hour time period. The apparatus in figure 2 shows a simple reaction vessel containing the catalysts and extract that are plumbed to a separate collection vessel. The extract is pumped out to the collection vessel by precipitation with 55 bar and 25℃.

Pure Δ9 THC

The final step described in the patent is the separation of CBD, Δ8 THC, and Δ9 THC. This is achieved by using a purification material/media commonly used in separation science – silica. As is described below in the chromatography, silica has a charge to it, that reacts with the molecules that are to be separated/purified from one another. Some molecules have a stronger interaction than others and therefore travel slower through the silica packed column. In this case, Mueller ends up with pure fractions of the three cannabinoids.

The silica used in the patent has an average size of 0.1mm, and is commonly used in separation science. Looking at figure 3, you will see that the CBD/THC mixture starts at the bottom of the column. When the supercritical CO2 is pumped into the system, the mixture travels up the column and begins to separate the mixture.

After the separation column, there are three collection vessels. As with the initial extraction, CBD, Δ8 THC, and Δ9 THC are separated out by precipitation. The precipitation occurs from dropping the P and T in steps from high to low. CBD is precipitated at 70 bar and 50℃. Δ8 THC is precipitated at 60 bar and 30℃, and Δ9 THC is precipitated at 55 bar and 25℃.

Separation of plant phytochemicals, terpenes, and cannabinoids

The first step in this patent produces pure THC/CBD while removing the plant alkaloids, flavonoids, and chlorophyll. It also removes terpenes.

Without good separation/purification, the terpene fraction can be contaminated with plant alkaloids, flavonoids, and chlorophyll. This is one of the inherent downsides to CO2 extractions compared to butane extractions; phytochemical contamination can be reduced in butane extractions by keeping conditions at sub-zero temperatures. In CO2 extractions, it can be reduced by not cranking the extractor into “hyperdrive” conditions.

Although the loss of terpenes is an inherent problem with supercritical CO2, it’s easiest to preserve terpenes by performing a subcritical run followed by supercritical run. This maximizes the yields, but is not explicitly described as the method used in this patent – it only suggests that the materials are extracted a second time.

Adsorbents

The use of adsorbents is illustrated by way of a packed column in figure 1. An adsorbent is a substance that attracts molecules, and the molecules subsequently adhere to the surface. Note that this is not the same as absorbing, where a molecule is taken into the structure of the substance. An adsorbant has a transient interaction with the molecules it attracts, where the molecules stay on the surface. An absorbant actually soaks up a molecule into its pores, rather than just staying on the surface.

Adsorbents are used in this patent to remove undesirable molecules, such as alkaloids, flavonoids, and chlorophyll. The adsorbents attract undesirable molecules. They adhere to the surface under sub and supercritical conditions, temporarily falling out of the supercritical solution, while the terpenes and cannabinoids stay in solution and pass on to the next section of the system. It is a clever way to remove the undesirables, but is relatively common in separations science.

The adsorbents are silica gel, diatomaceous earth, bentonite, bleaching earth, activated carbon, and mixtures of magnesium oxide and alumina zeolitic. Although it’s not explicitly said, there are two ways that the adsorbents can be used. First, you can pack the plant matter on top of a bed of adsorbents. The second method is to have secondary column in-line, downstream of the extraction chamber, that pulls out all undesirables. The second is much more clean and efficient, but requires additional equipment, and at these kinds of pressure ratings, stainless steel is not cheap.

Chromatography

A more subtle point, that may not be immediately apparent, is the application of chromatography. Chromatography is loosely defined as the separation of mixtures. In this case, the mixture to be separated is the undesirable plant phytochemicals and the desirable terpenes/cannabinoids. The main function of chromatography media (i.e. adsorbents and silica) is to allow some molecules to travel through the media faster than others.

When a molecule has a strong interaction with the chromatography/purification media, it travels slower. When a molecule has a weak interaction, or is repelled by the media, it travels faster. Imagine each phytochemical being a molecular magnet. For example THC and CBD are very weak magnets compared to alkaloids, flavonoids, and chlorophyll. In fact, the undesirable molecules are like strong magnets.

The way this works, is that the adsorbents are also like strong magnets, but have the opposite charge of the undesirable phytochemicals. Opposites attract, and the undesirable phytochemicals are strongly attracted to the adsorbent, while the desireable terpenes and cannabinoids have a weak attraction to the adsorbent. The weaker the interaction, the faster the molecules can travel through the media, and vice versa.

Extraction, purification, and resolution in supercritical extractions

Extraction is the first step in any purification scheme. The industry is currently focusing on whole plant extracts, without much purification beyond dewaxing. This patent utilizes purification in the process – this is how CBD, Δ8 THC, and Δ9 THC are separated from each other.

As described above, chromatography is the mechanism of separating molecules. This can be done by columns packed with silica, and other packing materials. In chemistry, the packing materials are called purification media. Whatever packing material is used, the purpose is to slow down some of the molecules in order to separate the ones you want from the ones you don’t want.

You can’t talk about chromatography without talking about resolution. Resolution can be simply defined as the amount of separation of two molecules in a purification process. Depending on what kind of packing materials used, you can separate groups of molecules from one another. Some packing materials work better for some types of molecules, and is a chore to find the right media when starting a new purification scheme.

When you put together the extraction step and follow it with purification steps, you can separate groups of molecules. The choice of using purification is one that is dependent on whether one wants a pure product.

The end product

One really needs to ask themselves how important purity is. The more pure of a product desired, the more effort that is required. Do you really want pure crystalline THC and CBD? If yes, this process may be right for you. If you just want an extract, you may want to consider these steps, but take what information is useful to you.

Again, a process is only useful if it is economically viable. The extra effort spent in making pure products is significant, but is a daily reality in the pharmaceutical industry. A point to consider is that the closer you get to a pure product, the higher the chance of losing your product due to mistakes.

Having spent years in chemical purifications, plenty of solvent and product have been lost to simple, avoidable mistakes. To avoid mistakes, clearly define your process before you begin. Always have a plan and think about what you’re going to do before you execute the plan. Be careful, take your time, take good notes, and always keep the safety of the consumer as your top priority.