Mastering HPLC for Cannabis and Hemp Analysis: A Q&A With Dr Lee Polite
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Cannabinoids. Pesticides. Terpenes. These are just three compound types on every cannabis analyst’s checklist. And as this list keeps growing, so does the need for accurate detection through high-performance liquid chromatography (HPLC) with robust method development.
Analytical Cannabis was delighted to recently host a webinar with Dr Lee Polite, president of Axion Analytical Labs, a chromatography training institute in Chicago, Illinois. Here we have summarized the answers Lee provided in response to live audience questions.
Roxanne Newman (RN): What are your thoughts on ethanol-water instead of acetonitrile (ACN) or methanol for the mobile phase?
Lee Polite (LP): Yeah, great question. I think it's a fantastic idea. We actually published a paper about a year ago on using ethanol as a mobile phase. Let me start with a general statement, ACN is probably the best mobile phase; methanol is not quite as good but almost as good. So, I say those two are almost interchangeable. Ethanol is about the same as methanol, although slightly not as good, as the viscosity of ethanol is higher so it’s going to generate slightly higher pressures. Certainly, [it has] a much higher pressure than ACN and a slightly higher pressure than methanol.
However, apart from that we found, ethanol had pretty similar separations to methanol, almost identical. So I would say it's not used very often. In fact, I've probably seen one other lab in the country use ethanol as a mobile phase.
But, as a scientist, I think there's no reason why you cannot use ethanol. My feeling is, make sure it's HPLC-grade; make sure there are no particulates in it, as that'll screw up your pump. Although it's not very common to use ethanol as mobile phase, we've had good luck with it, and I think it's perfectly fine.
RN: When working with plant extract samples, how can you know that you've got all the material off the column?
LP: That's a good question. Well, it's almost a philosophical question, we never really know if everything's come off the column. But the way I put it is, [if] we're going to stack the deck in our favour, we're going to run up to 100 percent of ACN at the end of our scouting run, our first gradient, with a ten minute hold time at the end of that. My feeling is, if I go to 100 percent ACN and I let it sit there for ten minutes, there are very few things that will stick to the columns. Very, very nonpolar things could, but, for the most part, I think we should get just about everything off.
If you suspect you have other stuff on the column, you could increase your column temperature. Most of us will run the LC column at 40°C, but, if you take that up to 60°C at 100 percent ACN, it makes it a much stronger solvent and good for column cleaning. But that's sort of getting philosophical, that's the best I can answer.
If you really, really want to know, and you have a total unknown, then we have to do what's called mass balance. You have to weigh the sample, inject it, quantify all the peaks, and then figure out what you have left over. In other words, you weigh a gram, and then you start trying to close that mass balance; was it water or was it something stuck on the column?
RN: How can we determine that it is time to change a column or that the column life is near to its end. Are guard columns worth using?
LP: Both [are] really good questions. First, let me deal with the guard column question. A guard column is a short version of your analyst column; it should contain the same packing material, the same stationary phase, the same size, same everything, but just a short version. And the idea is, if something bad is going to happen, let it happen to the cheap guard column and not the expensive analyst column. So that's the general concept of a guard column. And now I will make my weird statement: that I'm not a big fan of guard columns. Or, let me put it this way: on most of my applications, I do not use guard columns. [It’s] not that I'm against them. But here's how I determine their need.
If your sample is or might be dirty, then you should use a guard column. It's cheap insurance. If your sample is clean, and if you're running pure drinking water, then the guard column serves no purpose; you don't need one. So first determine what is clean versus dirty.
In HPLC, if something is a 100 percent soluble filter, everything through a half micron or point two-micron filter will damage a column. So, step one, make sure you got no particles on the column. If the sample is 100 percent in solution, and if it completely dissolves in methanol or ACN, then you can wash it off the column. So I don't worry about that kind of stuff that will never damage a column. If it's in solution, [it’s] not going to damage your column.
Now back to the excellent question: how do I know when it's time to change the column? Most people out there take a very random approach, X number of months, X number of injections. I think there's a much better approach, and that is, look at the resolution. I know a lot of people out there don't want to look at equations but forget about the equation, just look at the resolution number.
If your numbers are good, then your column is good. I'm a big fan of what I call performance-based measurements. So instead of saying, ‘throw your column after 1000 injections,’ I'd say throw away your column as soon as the resolution between your critical peaks drops below 1.5, or, if you're conservative, you could take 2.0.
So, I'm a big fan of that approach. As long as you still have good resolution, then the column is still good. If your columns are starting to look bad – if they're starting to be asymmetrical – you could wash the column again in 100 percent ACN or 100 percent methanol, take the temperature up to 60°C, and let it run for an hour. That should wash off just about anything that has stuck to your column.
RN: Have you seen any matrix interference at the 220nm detection wavelength, specifically in edibles?
LP: Not with distillates, but with edibles, yes. So, remember, chromatography is separation. If you're looking at a chocolate chip cookie, there's always a chance that a chemical in the matrix is going to come off on top of one of our cannabinoids.
The challenge about running at 220nm, which is a relatively low wavelength, is more compounds are detected. So, the answer is yes, we have seen coalition issues. As to how to deal with those, that's a little tougher question. Recently we've been playing around with the diode-array detector and using different wavelengths. So, in other words, monitor 220nm and see if you could, dependent on which cannabinoid you're looking at, monitor at 254nm, because you'll pick up a lot of cannabinoids there and you might not pick up that impurity. At 220nm, you'll start picking up organic acids. Anything in the organic acid in the sample itself will start to absorb UV light there.
RN: I'm having issues analysing THC distillate, which comes back as 75 percent THC. What's your view on sample prep?
LP: Oh, yeah, so sample prep. Rule number one, you have to dissolve 100 percent of the sample, you got to make sure everything's going in solution. Most of these samples, like THC and the cannabinoids, we find [to have] excellent solubility in methanol, ethanol, and ACN. Remember, when running HPLC, I would start with a 10th of a percent of a solution, so one milligram per ml solution. We don't need to make a really concentrated solution of this stuff. And that should get everything in the solution, and make sure we don't leave anything in the container.
Next thing to look at is the peak shape. THCA converts to THC, so we do have to watch out for that and also make sure you're within your linear range. In other words, if you calibrate your instrument using cannabinoid standards, and you sort of get up to 1000 ppm cannabinoids, you're not at the top of the calibration curve, but you're probably not too far from the top the calibration curve. Make sure your absorbance units don't go above 2000 milli-absorbance units (mAU), as you’ll be losing linearity of the detector.
Make sure the peak is not too big, I guess is a takeaway from that.
RN: How do you handle ion suppression?
LP: Yeah, ion suppression, that's a good one. So, in general, when we do reverse phase chromatography, we like all our compounds to be neutral, as in not ionized; acids will ionize at a high pH and bases will ionize at low pH, so ion suppression means we set the pH to keep everything suppressed.
I run almost everything at pH 2, this is my best kept secret, although I guess it's not a secret anymore. So, at pH 2, I will suppress every organic acid that could be suppressed. With basic compounds, they will ionize and be positively charged, but I'm also a big fan of base-deactivated columns.
So, if you buy a good base-deactivated column, that has been end-capped or sterically protected, then having an ionized base is not a problem at all. My favourite mobile phase is phosphoric acid; about 0.3 percent phosphoric acid in water. I put that in all my water bottles. My other little trick is I add 5 percent methanol to all my water bottles. That's for microbial growth that keeps the algae from growing.
RN: Are many or most of typical cannabinoid terpenes able to be analyzed by HPLC or does their volatility limit them to GC?
LP: Oh, yeah, that's a good question. I would say most of them can be done by HPLC. In deciding between using HPLC and GC, they're very complimentary techniques. So, GC is used for anything that is volatile; if you could vaporize it, we can definitely do it by GC. Liquid chromatography is used for anything that is soluble; if you dissolve it, we could do it by LC. Now there's a lot of things that go both ways.
Even though the cannabinoids and the terpenes are volatile, which means they can be done by GC, it doesn't mean they cannot be done by LC. To me, the trick in LC is step one, can I dissolve it? And then question number two, how can I detect it? Can I use my UV detector? So, if we can answer yes to those two questions, we can certainly do the terpenes and cannabinoids by HPLC and many of them by GC as well. Just not the acids by GC, because those decarboxylate.