Investigating the effect of polarity of stationary and mobile phases on retention of cannabinoids in normal phase liquid chromatography
Analytical and Bioanalytical Chemistry
https://doi.org/10.1007/s00216-021-03862-y
RESEARCH PAPER
Investigating the effect of polarity of stationary and mobile phases
on retention of cannabinoids in normal phase liquid
chromatography
Chiara De Luca1 · Alessandro Buratti1 · Yannick Krauke2 · Svea Stephan2 · Kate Monks2 · Virginia Brighenti3 ·
Federica Pellati3 · Alberto Cavazzini1 · Martina Catani1 · Simona Felletti1,2
Received: 15 November 2021 / Revised: 10 December 2021 / Accepted: 20 December 2021
© The Author(s) 2022
Abstract
This work reports about a screening of four adsorbents with different polarity employed for the separation of the main
phytocannabinoids contained in Cannabis sativa L., under normal phase liquid chromatography (NPLC). The effect of
polarity and type of interaction mechanisms of the adsorbents (namely Si-, CN-, Diol-, and NH2 -based SPs) on retention has
been investigated under a variety of conditions either by using different combinations of apolar solvents (heptane or hexane)
and alcohols (ethanol or isopropanol). The columns have also been employed for the separation of a real cannabis sample.
Keywords Cannabinoids · Cannabis sativa L. · HPLC · Hemp · Normal phase · Polar-bonded phases
Introduction
In the last years, there has been an increased interest around
the potential of cannabis-based products for medical and
nutraceutical purposes. Cannabis sativa L., in particular,
contains a large number of bioactive compounds, including
flavonoids, terpenoids and, most importantly, cannabinoids,
among which cannabidiol (CBD) and tetrahydrocannabinol
(9 -THC) are the most popular and investigated ones.
Published in the topical collection featuring Promising EarlyCareer (Bio)Analytical Researchers with guest editors Antje
J. Baeumner, Marı́a C. Moreno-Bondi, Sabine Szunerits, and
Qiuquan Wang.
Martina Catani
Simona Felletti
1
Department of Chemical, Pharmaceutical and Agricultural
Sciences, University of Ferrara, via L. Borsari 46, 44121
Ferrara, Italy
2
KNAUER Wissenschaftliche Geräte GmbH, Hegauer Weg 38,
14163, Berlin, Germany
3
Department of Life Sciences, University of Modena
and Reggio Emilia, Via G. Campi 103, Modena 41125, Italy
These two cannabinoids are not directly synthesized by
the plant but they are produced after exposure to heat and
light of their acid precursors (cannabidiolic acid, CBDA,
and tetrahydrocannabinolic acid, THCA, respectively) [1–
3], which represent the most abundant compounds naturally
occurring in Cannabis sativa L..
9 -THC is known for its psychotropic effect. Its
assumption, therefore, underlies strict regulations in many
Countries. On the other hand, CBD does not get people
high and it is not responsible for intoxicating effects. For
this reason, it is one of the most studied and promising
bioactive cannabinoids. Ongoing research is focused on the
potential of CBD for the treatment of cancer, pain and
many neurological diseases [4]. In addition, it possesses
anti-inflammatory, anti-oxidant and anti-epilectics agents
[5–8]. For the reasons above, the demand of pure CBD is
continuously increasing and cannabis industry is demanding
for efficient methods to separate and purify CBD from other
components. However, purification of CBD from cannabis
extracts could be challenging due to the complexity
of the matrix, which includes other chemically similar
cannabinoids, in addition to terpenes, waxes, etc. [9].
Preparative liquid chromatography is by far the most
widely applied method in industry for the purification
of single components from complex mixtures. The most
important advantage of this technique is the great versatility
that can be modulated through the combination of different
adsorbents and eluents to achieve the separation of a wide
C. De Luca et al.
range of compounds [10–13]. Several studies have already
demonstrated that reversed-phase liquid chromatography
(RPLC) can be efficiently applied for the separation
and simultaneous quantification of a large number of
cannabinoids [3, 14–20], at the point that both Dutch
and German Pharmacopoeias report HPLC-UV as the
official method for potency testing [21, 22]. Conversely,
no fundamental studies about the employment of normal
phase liquid chromatography (NPLC) for the separation
of cannabinoids have been published so far with the
exception of some works investigating the potential of
NPLC for the chiral separation of cannabinoids on chiral
stationary phases and some technical notes by Companies
[19, 23–29]. On the opposite, being based on intrinsically
different retention mechanisms compared to RPLC, NPLC
might provide higher selectivity and resolution in some
cases [30]. For instance when poor resolution of analytes
under RP conditions is observed (e.g., the separation of
the critical pair CBD-CBG [19]) or when impurities are
more hydrophobic than the target analyte (in these cases,
they are very strongly retained in RPLC, while could
be quickly eluted in NPLC [31]) the employment of
NPLC could be advantageous. Moreover, the use of apolar
solvents facilitates sample preparation, especially of real
samples. Indeed, in hexane or heptane the annoying issue
of precipitation of apolar compounds (such as terpenes,
abundandtly present in real samples of cannabis) is avoided.
At the same time, sample solubility is increased in
apolar solvents and therefore also column loading, while
solvent removal from purified fractions is easier, which
are both very important aspects from both a preparative
and environmental viewpoint. Concerning sustainability
of organic solvents, heptane and acetonitrile (which is
commonly used in RPLC) both belong to the same class
of “problematic” solvents [32], therefore the environmental
impact of the two methods is almost the same. Finally, the
use of low-viscosity solvents is less demanding in terms
of pump back-pressure allowing for higher flow rates (i.e.,
faster runs).
Retention in NPLC has been usually described by
the displacement model of retention for liquid-solid
chromatography [33]. Briefly, the surface of the stationary
phase is covered by a monolayer of solvent molecules that
have to be displaced by the analyte molecule in order to
be retained. In other words, solute and solvent molecules
compete for adsorption on a limited number of adsorption
sites. The understanding (and the prediction) of stationary
phase selectivity in NPLC is a very complicated topic
[30, 33–42]. The type of functional groups present on
the stationary phase but also the nature of mobile phase
modifier have a great effect on selectivity. The importance
of hydrogen bonding has been recognized as one of
pivotal aspects to be considered to understand retention and
selectivity in NPLC [33, 34, 43–45].
Bare silica (Si) bears unbonded silanol groups (SiOH) on the surface of the particles that are strong
proton donors. They can interact via hydrogen bondingtype interactions with hydrogen bond acceptor groups (i.e.,
molecules with available (...truncated)
