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From The Lab

HPLC Column Selection for Cannabis Chromatographers

By Danielle Mackowsky
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If your laboratory utilizes an HPLC system for cannabinoid and pesticide analysis, it can be a daunting task to select a stationary phase that is both practical and sufficient for the separation at hand. Typically, when developing a new method, an analyst will either evaluate a column they already have in house or seek out a referenced phase/dimension in the literature before exploring other available alternatives.

Tetrahydrocannabinol (THC)
Chemical structure of Tetrahydrocannabinol (THC)

A C18 phase is an excellent first choice for non-polar or slightly polar compounds. If the analyte in question has a minimum ratio of three carbon atoms for every heteroatom, it will be sufficiently retained on this phase. THC and other relative cannabinoids are prime candidates for separation via C18 due to their non-polar nature and structural components.

In addition to a universal C18 phase, alternative selectivity options do exist for laboratories concerned with the analysis of cannabinoid content. Another prevalent column choice features an aromatic or poly-aromatic stationary phase. Compatible with highly aqueous mobile phases, aromatic and poly-aromatic columns primarily rely on hydrophobic and π-π interactions as their main analyte retention mechanisms. Poly-aromatic phases provide enhanced retention and are more hydrophobic when compared to a single phenyl ring structure. While C18 phases are not ideal for resolving structural isomers, poly-aromatic columns are capable of separating these ring-based compounds. Chromatographers with a background in forensic analysis may be very familiar with this type of HPLC column due to its extensive use in drug testing applications.

Chemical structure of chlormequat, a hazardous polar pesticide commonly banned for use in cannabis cultivation
Chemical structure of chlormequat, a hazardous polar pesticide commonly banned for use in cannabis cultivation

Besides cannabinoid content, many cannabis scientists are equally concerned with accurate quantitation of pesticides within a given sample. Many pesticides that have found themselves on regulatory lists in states such as Massachusetts, Washington or Nevada are extremely polar. In order to increase retention of these compounds, and thus improve your overall chromatographic method, it can be extremely advantageous to select a column that allows you to start your gradient at 100% aqueous mobile phase. An aqueous or polar modified C18 column contains an embedded polar group, polar side chain or polar end-capping to allow for separation of polar compounds, while still retaining and resolving non-polar analytes. For laboratories that necessitate the use of only one analytical column, an aqueous C18 phase will allow for separation of monitored pesticides without compromising the quality of cannabinoid data produced.

One must also take into account column length, pore size and particle size when purchasing a column. For the purposes of any cannabis related analysis, a pore size of 100-120Å will suffice. Larger pore columns are typically reserved for large peptides, proteins and polymers. Depending on the sensitivity and resolution needed within your laboratory, particle size can range from 1.8-5um, with the highest sensitivity and resolution coming from the smaller particle size. Core shell technology is also a popular option for laboratories who want to keep the pressure of their HPLC system low, without sacrificing any quality of their resolution. Column lengths of 50 or 100 mm are common for chromatographers who want to achieve sufficient sample separation while keeping their run times relatively short.UCTcolumns

Regardless of the HPLC phase selected, it is very important that a guard cartridge is also used. Guard cartridges are traditionally the same phase and particle size of the HPLC column choice and help to prolong analytical column life. They provide additional sample clean up and are widely recommended by the majority of chromatography experts. Upon reviewing one’s options for HPLC phases and acquiring the necessary guard column, your cannabis laboratory will be ready to get the most out of your HPLC system for your analysis needs.

amandarigdon
The Practical Chemist

Easy Ways to Generate Scientifically Sound Data

By Amanda Rigdon
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amandarigdon

I have been working with the chemical analysis side of the cannabis industry for about six years, and I have seen tremendous scientific growth on the part of cannabis labs over that time. Based on conversations with labs and the presentations and forums held at cannabis analytical conferences, I have seen the cannabis analytical industry move from asking, “how do we do this analysis?” to asking “how do we do this analysis right?” This change of focus represents a milestone in the cannabis industry; it means the industry is growing up. Growing up is not always easy, and that is being reflected now in a new focus on understanding and addressing key issues such as pesticides in cannabis products, and asking important questions about how regulation of cannabis labs will occur.

While sometimes painful, growth is always good. To support this evolution, we are now focusing on the contribution that laboratories make to the safety of the cannabis consumer through the generation of quality data. Much of this focus has been on ensuring scientifically sound data through regulation. But Restek is neither a regulatory nor an accrediting body. Restek is dedicated to helping analytical chemists in all industries and regulatory environments produce scientifically sound data through education, technical support and expert advice regarding instrumentation and supplies. I have the privilege of supporting the cannabis analytical testing industry with this goal in mind, which is why I decided to write a regular column detailing simple ways analytical laboratories can improve the quality of their chromatographic data right now, in ways that are easy to implement and are cost effective.

Anyone with an instrument can perform chromatographic analysis and generate data. Even though results are generated, these results may not be valid. At the cannabis industry’s current state, no burden of proof is placed on the analytical laboratory regarding the validity of its results, and there are few gatekeepers between those results and the consumer who is making decisions based on them. Even though some chromatographic instruments are super fancy and expensive, the fact is that every chromatographic instrument – regardless of whether it costs ten thousand or a million dollars – is designed to spit out a number. It is up to the chemist to ensure that number is valid.

In the first couple of paragraphs of this article, I used terms to describe ‘good’ data like ‘scientifically-sound’ or ‘quality’, but at the end of the day, the definition of ‘good’ data is valid data. If you take the literal meaning, valid data is justifiable, logically correct data. Many of the laboratories I have had the pleasure of working with over the years are genuinely dedicated to the production of valid results, but they also need to minimize costs in order to remain competitive. The good news is that laboratories can generate valid scientific results without breaking the bank.

In each of my future articles, I will focus on one aspect of valid data generation, such as calibration and internal standards, explore it in practical detail and go over how that aspect can be applied to common cannabis analyses. The techniques I will be writing about are applied in many other industries, both regulated and non-regulated, so regardless of where the regulations in your state end up, you can already have a head start on the analytical portion of compliance. That means you have more time to focus on the inevitable paperwork portion of regulatory compliance – lucky you! Stay tuned for my next column on instrument calibration, which is the foundation for producing quality data. I think it will be the start of a really good series and I am looking forward to writing it.