Jeff Tries Cannabis Part 2: HPLC Method Development

Blog 2: Effect of Different Mobile Phase Modifiers.

Guest Author: Jeff Layne

Jeff's head

Welcome back to my series on method development for potency testing of naturally-occurring cannabinoids. In the previous article, I had done preliminary gradient screening of our 12 cannabinoids using a 100 x 4.6 mm Kinetex® C18 column and a series of water, acetonitrile, and 0.1% formic acid gradients. Using a 75-100% gradient profile, I was able to partially resolve 11 of the 12 target cannabinoids. However, trying a different acid might result in a change in selectivity that reveals the location of the 12th peak.


As you can see from the results in Figure 1, there was surprisingly vMethod Development Cannabis testing Phenomenex ery little difference in the behavior of the different modifiers that we evaluated (formic acid, phosphoric acid, TFA, and plain water). In fact, I would say that it made no difference at all which of the acids were added to the mobile phases. However, based upon the chromatography when using water and acetonitrile without any modifier (Fig. 1d), you would want to add something to bring the pH down. By not injecting the individuals at this point, it is unknown which analytes moved around in the plain water chromatogram, but it is safe to assume it is most likely the acidic compounds that are moving around as they are most likely to be affected by changes in mobile phase pH (and in the protonation of any residual silanol groups).

While this data indicates that using different acids is not a useful tool in modifying the retention behavior of our target cannabinoids, and will also not help resolve the mysterious 12th cannabinoid peak, it does not mean that this was a useless exercise. In fact, I think it is exceptionally useful because it could be used later when dealing with different sample matrices. For instance, one person may be trying to determine potency in the raw plan materials, while another may be interested in quantifying the cannabinoids present in a food product.

Each matrix will have its own unique set of potential interferences. The different acids could be used as a method development tool to attempt to move matrix interferences away from the target cannabinoids of interest. As the acid modifier changes, we know the cannabinoids will stay in the same place, but hopefully the different acids can be used to move the different matrix interferences away from the peaks of interest.

What other types of mobile phases would you have evaluated?

Figure 1. Effect of different mobile phase modifiers (each at 0.1% in water and acetonitrile; gradient 75-100% B in 10 min).
a. Formic acid
Formic Acid mobile phase Cannabis
b. Phosphoric acid
Phosphoric Acid mobile phase Cannabis
c. Trifluoroacetic acid (TFA)
Trifluoroacetic acid (TFA) mobile phase cannabis
d. Water and acetonitrile without any modifier
Water and acetonitrile in cannabis

This might be a good stopping point for right now. So far, we’ve done some preliminary gradient screening and looked at different acidic modifiers. In my next article, I’ll continue with method development in the search for the mysterious 12th analyte. There are still many options, including further optimizing our gradient, looking at the effect of temperature, and evaluating different flow rates. Don’t forget that we haven’t even touched on alternative stationary phases of column dimensions yet either. Your comments, feedback, and suggestions are welcome!

See you in the next article!

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