7 highly important factors for cannabinoid isolation

It can be challenging to zig zag through all the science involved in cannabis processing. But I’ve got an ounce of good news for you. In this post, I try to help you out by spilling the tea on how optimize one of the most essential steps of the cannabis processing workflow, cannabinoid purification by chromatography. Check out seven parameters that I find to be crucial for the success of your CBD, THC and terpene purifications.

I’ve written about my love-hate relationships with DIY videos before in a previous chromatography post . I now stumbled upon two guys who make videos of themselves trying out DIY videos and I had to have a good laugh. In one video, they attempt to replicate a recipe for cloud bread, fail to beat their egg whites to stiff peaks and just keep adding corn starch in a fruitless effort to rescue their bread. The final baked product looked less like a cloud and more like a pancake with more cracks on it than the Moon.

Well, it was pretty clear the recipe was going to be doomed, even to the two of them. When one part of your workflow, getting the stiff peaks, fails, the rest of the procedure won’t turn out as planned either.

The same is true for any multi-step process anywhere. Consider cannabis processing. You need to grow, harvest, extract, concentrate, purify, control quality and if any of these steps fails or is performed sub-optimally, the rest of the downstream processing is inevitably affected negatively.

So I am here to save the day, or at least your cannabis product, by giving you some tips on how to optimize the cannabinoid purification part of the workflow.

1. Method development – Establish your chromatography method on a small scale first and only upscale once you have all parameters optimized. Take a look at the chart below to see typical stationary phases, mobile phases, flow rates and gradients used for cannabinoid separation

Eluent A (weak solvent)Water
Eluent B (strong solvent)Ethanol
Flash cartridge12 g FlashPure EcoRex C18
Flow rate30 mL/min
t (min)Gradient Type%B
0 - 6.4Linear70 - 90
6.4Step100
6.4 - 16Hold100

A typical chromatography run for purifying cannabinoids has values as follows:

 General column volume (CV)
Equilibration3-6
Injection volumemax. 0.1
Separation3-6
Rinsing (strong solvent)2-3

Afterwards, ideally, your chromatogram of how CBD, THC and THCA separate should look like the one below:

chromatography, cannabinoids, THC, CBD, THCA

To upscale, use the same solvents, stationary phase, gradient and linear velocity.

2. Pressure – The particle size and radius are the factors that have the most substantial influence on the pressure. Be aware that every cartridge has a maximal pressure limit that you should not exceed. If you use higher back pressures, you need to lower the flow rate, due to longer run times.

3. Sample loading – Try to keep the sample volume as low as possible. I would not recommend exceeding 0.1 column volumes. Keep in mind that viscous samples and obstructed filters can decrease the injection flow. Make sure you clean or change your solvent filters regularly.

4. Use ELSD as your detection method – Most chromatography systems are equipped with UV detectors . But consider the advantages that ELSD can bring to effectively separating cannabinoids. ELSD can detect a wide range of entities, including non-chromophore compounds, which are impossible to analyse by UV detectors alone. Importantly, this includes terpenes of cannabis.

5. Stationary phase selection – C-18 is the most commonly used stationary phase to separate cannabis extracts with a chromatography system . Different particle sizes of this stationary phase are available. When you use smaller particle sizes, you can achieve higher loading capacity and higher resolution. However, with smaller particle sizes, you also generate higher back pressure, so you would be forced to reduce the flow rate and increase the run time.

6. Loading capacity – The loading capacity depends on the type of stationary phase and on the size of cartridge . For example, a C18 50 µm can be loaded with cannabinoids as follows:

Cartridge sizeMax loadability
1.5 kg1.5 - 37.5 g
3 kg3 - 75 g
5 kg5 - 125 g

7. Fraction collection – This is an important factor to consider if you suffer from a lower resolution during cannabinoid separation. If you focus the collection at the point without overlapping, you can still achieve high purity. You can further purify contaminated fractions either by re-injection or by re-crystallization.

I hope you found this information highly useful. But there is even more you can learn on the topic with a concise free poster on tips and tricks for cannabinoid separations . Regardless if your knowledge is green or you are an expert cannabis scientist, there is always something new to discover. Keep dropping by the blog for more useful ideas on how to perfect your chromatography runs!

Till next time,

The Signature of Bart Denoulet at Bart's Blog