Journal of Chromatography A, 1599 (2019) 196–202

Contents lists available at ScienceDirect

Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Rapid isolation of acidic cannabinoids from Cannabis sativa L. using

pH-zone-refining centrifugal partition chromatography
Johannes R. Popp a,b,1 , Eleftherios A. Petrakis a,1 , Apostolis Angelis a , Maria Halabalaki a ,
Günther K. Bonn c , Hermann Stuppner b , Leandros A. Skaltsounis a,∗
a
Department of Pharmacognosy and Natural Products Chemistry, Faculty of Pharmacy, National and Kapodistrian University of Athens, Panepistimiopolis
Zografou, 15771 Athens, Greece
b
Department of Pharmacognosy, Institute of Pharmacy, Center for Molecular Biosciences (CMBI), University of Innsbruck, Innrain 80-82, 6020 Innsbruck,
Austria
c
Institute of Analytical Chemistry and Radiochemistry, Center for Chemistry and Biomedicine (CCB), University of Innsbruck, Innrain 80-82, 6020
Innsbruck, Austria

a r t i c l e i n f o a b s t r a c t

Article history: This work introduces an effective methodology for the isolation of acidic cannabinoids from fiber-type
Received 11 December 2018 Cannabis sativa L. Supercritical fluid extraction (SFE) was initially employed to obtain an enriched extract
Received in revised form 23 March 2019 of acidic cannabinoids. Subsequently, fractionation was performed by using centrifugal partition chro-
Accepted 17 April 2019
matography (CPC) with the pH-zone-refining method. The biphasic solvent system that was selected
Available online 18 April 2019
consisted of n-hexane/ethyl acetate/ethanol/water 8:2:5:5 (v/v/v/v). Trifluoroacetic acid was added as
retainer in the organic stationary phase, while triethylamine was used as eluter in the aqueous mobile
Keywords:
phase. The most promising CPC fractions containing cannabidiolic acid (CBDA) and cannabidivarinic acid
Cannabidiolic acid (CBDA)
Cannabidiol (CBD)
(CBDVA) were further purified by liquid-liquid extraction. Following this procedure, 1.86 g of CBDA (>85%)
Cannabidivarinic acid (CBDVA) were recovered from 9 g of extract, with 1.08 g thereof having a purity of more than 95%, as determined by
Industrial hemp HPLC-PDA analysis. Moreover, 91 mg of CBDVA with greater than 85% purity were obtained. This method-
Preparative isolation ology can be efficiently used for the large-scale purification of CBDA and after minor modifications could
Centrifugal partition chromatography (CPC) be readily adaptable for the isolation of other acidic cannabinoids, based on their ionizable character.
© 2019 Elsevier B.V. All rights reserved.

1. Introduction The cannabinoids 9 -THC and CBD are commonly present in

C. sativa extracts, but are naturally in their acidic forms. Acidic
The plant Cannabis sativa L. is the natural source of about 120 cannabinoids are known as the biogenetic precursors of the corre-
cannabinoids [1], which are also referred to as phytocannabinoids, sponding neutral ones and, as suggested by Izzo et al. [8] as well as
to emphasize their botanical origin [2]. Cannabinoids constitute a by Cascio and Pertwee [9], may exert very weak or no psychotropic
distinct class of meroterpenoids, or more specifically, terpenophe- effects. In the presence of light or heat, neutral cannabinoids are
nolic compounds that have rendered C. sativa as one of the formed due to the decarboxylation of their acidic precursors [10].
most controversial plants throughout history [3]. In recent years, However, the pharmacological effects of heated C. sativa and related
though, numerous therapeutic effects of C. sativa, largely associ- extracts containing the neutral ones may not be enhanced, com-
ated with cannabinoids, are increasingly reported [4–7]. Diverse pared with the unheated extracts. The latter usually contain higher
pharmaceutical properties of the principal neutral cannabinoids, amounts of acidic cannabinoids and can be accompanied with bet-
9 -tetrahydrocannabinol (9 -THC) and cannabidiol (CBD), have ter tolerability and a lower rate of adverse effects [11].
been well documented [5–8]. CBD is mainly derived from fiber-type Cannabidiolic acid (CBDA) was the first acidic cannabinoid to be
C. sativa (industrial hemp or hemp) and, in contrast to 9 -THC, it isolated in 1955 [12]. Although it comprises the major cannabinoid
is not psychotropic. of fiber-type C. sativa, CBD remains its main pharmacological agent
and only a limited number of studies have investigated whether
CBDA itself is biologically active. CBDA has been reported to possess
∗ Corresponding author at: Department of Pharmacognosy and Natural Products antibacterial properties [13] and is a promising compound for the
Chemistry, Faculty of Pharmacy, National and Kapodistrian University of Athens, treatment of vomiting and nausea, including both acute and antici-
Panepistimiopolis Zografou, 15771 Athens, Greece. patory nausea [14–17]. In addition, this acidic cannabinoid has been
E-mail address: [email protected] (L.A. Skaltsounis). shown to exhibit anti-hyperalgesia and anti-inflammatory effects
1
Authors with equal contribution.

https://doi.org/10.1016/j.chroma.2019.04.048
0021-9673/© 2019 Elsevier B.V. All rights reserved.
 J.R. Popp et al. / J. Chromatogr. A 1599 (2019) 196–202 197

[18–20]. Because it is an inhibitor of cancer-related proteins, CBDA temperature and maintained in the absence of light. For perform-
may also inhibit breast cancer cell migration [21–23]. ing CPC separations, solvents of analytical grade, i.e. ethyl acetate
Considering the growing interest in the biological activ- (EtOAc), ethanol (EtOH), and n-hexane (n-Hex), were purchased
ities of CBDA and other acidic cannabinoids, such as 9 - from Carlo Erba Reactifs SDS (Val de Reuil, France). Water, acetoni-
tetrahydrocannabinolic acid (9 -THCA) or cannabigerolic acid trile (ACN), and formic acid of HPLC grade (Carlo Erba) were used for
(CBGA) [8,9], efficient methodologies for their isolation and purifi- HPLC analysis. For LC–MS analysis, acetonitrile and formic acid of
cation are required for producing large amounts of high purity LC–MS grade were acquired from Fisher Scientific (Leicestershire,
grade material. Flash chromatography was recently applied for UK), while ultrapure water was obtained from a Milli-Q® purifi-
the isolation of THCA [24]. The procedure was carried out using cation system (Merck Millipore, Darmstadt, Germany). Methanol
either normal-phase or reversed-phase chromatography. Although (MeOH), dichloromethane (DCM), hydrochloric acid, and sulfuric
a high purity grade was achieved in both cases (≥98.8%), the load- acid (>95%) were obtained from Fisher Scientific, while vanillin
ing capacity for the sample is limited and thus only milligrams of (98%), trifluoroacetic acid (TFA), and triethylamine (TEA) were pur-
the acidic cannabinoid were obtained. chased from Sigma-Aldrich (Steinheim, Germany).
Previous studies have indicated the potential of centrifugal
partition chromatography (CPC) for the large-scale isolation of 2.2. Supercritical fluid extraction (SFE)
cannabinoids [25,26]. CPC or hydrostatic counter-current chro-
matography is a solid support-free separation technique that Preliminary experiments were carried out using a lab-scale
permits easy scaling up. The distribution of solutes is carried out SFE apparatus (SEPAREX, Champigneulles, France), with a view to
between at least two immiscible liquid phases, based on their par- assess the effect of ethanol as a co-solvent at different propor-
tition coefficients. The stationary phase is maintained inside the tions (i.e. 0-5-10-20% w/w). Each experiment was performed for
column cells by centrifugal force and the mobile phase is contin- 2 h, by filling the extraction vessel (100 mL) with 20 g of C. sativa
uously eluted at high flow rates [27]. The capability of CPC for the dried material. It was shown that the extract obtained using 100%
preparative isolation of acidic cannabinoids has been successfully CO2 was the most concentrated in CBDA. In addition, the CO2 flow
demonstrated [25,26]. Nevertheless, the method proposed requires rate was adjusted in order to minimize solvent consumption while
fractionation of the n-hexane extract prior to separation, to perform retaining the extraction yield. Afterwards, the C. sativa dried mate-
CPC exclusively with the fraction of acidic cannabinoids. This pro- rial (ca. 300 g) was submitted to supercritical fluid extraction using
cess yielded CBDA with a purity of 90.2%, while the purity of the a pilot-scale SFE 1–2 No. 4218 apparatus (SEPAREX) consisting of a
acidic cannabinoids isolated from another C. sativa extract, namely CO2 tank, a liquid CO2 pump (up to 10 kg/h capacity), two directly
THCA and CBGA, was less than 95% as determined by gas chro- connected extraction vessels (i.e. 1 & 2 L), three separators (200 mL
matography (GC) [25]. capacity each), a co-solvent pump, and a cooling system using gly-
The separation of ionizable analytes may effectively take place col. The extraction was carried out within a stainless steel basket
using CPC in pH-zone-refining mode [27]. This elution mode can be placed in the 2-L tubular extractor. The pressure in the extraction
selective for separating organic acids according to their respective vessels was kept constantly at 15 MPa, while the pressure in the
pKa ’s and hydrophobicity [28,29]. The pH-zone-refining method- separators compartment was held at 4 MPa. The extraction and sep-
ology was first introduced in 1994 [28], and since then has been aration temperatures were set at 35 ◦ C and 30 ◦ C, respectively. The
extensively reported, especially for the preparative separation of extraction was static with continuous recycling of the CO2 gas at a
amino acids and peptides [30], acidic analytes such as cichoric flow rate of 5 kg/h. The extract was partially collected every 40 min
acid [31], caffeoylquinic acid isomers [32], triterpene acids [33] and the whole SFE procedure lasted approximately 15 h. Finally,
and acidic triterpenoid saponins [34], basic analytes including alka- the extract was submitted to lyophilization and the total yield was
loids [35–37], and phenolic compounds [37,38]. The key advantages estimated as % (w/w) of the dried plant material.
of this methodology include enhanced loading capacity, minimum
overlap of rectangular compound peaks and concentration of frac- 2.3. Gas chromatography - mass spectrometry (GC–MS)
tions near saturation level [30].
The present study introduces a newly developed method for the For GC–MS analysis, samples were diluted in DCM (1 mg/mL)
preparative isolation of acidic cannabinoids such as CBDA. Herein, and an aliquot of 1 ␮L was injected in splitless mode on a Finnigan
the use of CPC in pH-zone-refining mode was investigated for the Trace GC Ultra 2000 apparatus (Thermo Electron Corporation, USA),
first time to isolate CBDA from the hemp extract obtained by super- equipped with an AI 3000 autosampler. The GC system was cou-
critical fluid extraction (SFE). Following CPC fractionation, a simple pled with a Finnigan Trace DSQ mass selective detector, operating
liquid-liquid extraction step was performed for further purifica- in Electron Impact (EI) mode. Xcalibur 2.0.7 software (Thermo Sci-
tion. This approach indicated high recovery rates of purified CBDA, entific) was used for the operation of the system as well as for data
while an enriched fraction of cannabidivarinic acid (CBDVA) was handling and processing. The injector and detector temperatures
also obtained. were set at 220 and 280 ◦ C, respectively, while the carrier gas was
helium at a flow rate of 1 mL/min. The capillary column used was
2. Materials and methods a Trace TR-5MS (Thermo Scientific, USA) column (30 m × 0.25 mm;
film thickness 0.25 ␮m) and the analysis was carried out according
2.1. Plant material and reagents to Hazekamp et al. [40], using a temperature programme starting
from 100 ◦ C and reaching 280 ◦ C with a rate of 10 ◦ C/min. The final
Aerial parts of an EU-approved fiber-type C. sativa variety temperature was held isothermally for 12 min and the total run
(’Fedora 17’) [39], harvested during the flowering period in October time was 30 min.
2016, were provided by a registered producer in Boeotia (central
Greece). The plant material was certified for a THC content not 2.4. Determination of the partition coefficients (K)
exceeding 0.2% (w/w) and a voucher specimen has been deposited
in the Herbarium of the Department of Pharmacognosy and Natu- Approximately 10 mg of the C. sativa extract were weighed into
ral Products Chemistry at University of Athens (LSCs-16-01). After three separate 10-mL test tubes and subsequently 4 mL of the n-
air-drying at 25 ◦ C, the hemp sample was ground in a mill until a Hex/EtOAc/EtOH/H2 O 8:2:5:5 (v/v/v/v) system were added to each
fine powder was obtained. Finally, it was placed in storage at room tube. For two of them, the pH was adjusted to 2 and 10 by using
198 J.R. Popp et al. / J. Chromatogr. A 1599 (2019) 196–202

TFA and TEA, respectively. Then, the contents of each tube were (Thermo Scientific, San Jose, CA, USA), equipped with a P4000
thoroughly mixed and allowed to settle at room temperature. In binary pump, an AS3000 autosampler, a column thermostat, and a
all cases, the settling time was short (<30 s) and two clear layers UV6000LP photodiode array detector (PDA). ChromQuest 5.0 soft-
were formed. An aliquot (300 ␮L) of each phase was used for HPLC- ware (Thermo Scientific) was used for the operation of the system
PDA analysis, after solvent evaporation and reconstitution in 1 mL and data processing. Samples were injected into a reversed-phase
ACN/MeOH 80:20 (v/v). The partition coefficients (K) in solvent Ascentis C18 (Supelco, Sigma-Aldrich, USA) column (150 × 2.1 mm,
systems were calculated using the following formula: 3 ␮m) and the compounds were eluted using a gradient mode of
0.1% formic acid in water (solvent A) and 0.1% formic acid in ace-
AU
K= (1) tonitrile (solvent B). Separation was achieved under the gradient
AL
conditions suggested by Giese et al. [42] with slight modifications,
with AU and AL being the peak area of the target compound in the that is: 66% B for 9 min, followed by a linear gradient to 95% B
upper and lower phase, respectively. over 4.5 min; isocratic for 1 min and then from 95 to 66% B in
4.5 min, followed by isocratic conditioning at 66% B for 6 min. Total
2.5. Centrifugal partition chromatography (CPC) run time was 25 min and UV spectra were recorded from 200 to
400 nm, while the PDA was set at 220, 275 and 324 nm for all com-
The fractionation of the C. sativa extract was performed by using pounds. The column temperature was kept at 25 ◦ C. The flow rate
an FCPC1000® instrument (Rousselet-Robatel Kromaton, Annonay, was 0.45 mL/min and the injection volume was 5 ␮L. The CBDA con-
France) equipped with a preparative column of 1 L (actual volume tent in the SFE-CO2 extract was calculated by a 5-point calibration
955 mL [41]) (Angers, France), a Prep 36 LabAlliance dual piston curve using the isolated CBDA (>95%) at a concentration range of
pump (State College, PA, USA), and a Flash 14 DAD detector (ECOM, 25–500 ␮g/mL in ACN (R2 = 0.9992).
Prague, Czech Republic) controlled by Clarity software. Three wave-
lengths were chosen to detect eluted compounds; 220, 275, and
2.8. Liquid chromatography – high resolution mass spectrometry
324 nm. The extract was injected into the system through a 50-
(LC-HRMS)
mL loop and eluted fractions were collected by a Büchi B-684
fraction collector (Flawil, Switzerland). CPC fractionation with the
LC-HRMS analysis was performed using an Accela ultra-high
pH-zone refining method was performed using the biphasic sys-
performance liquid chromatography (UHPLC) system hyphenated
tem n-Hex/EtOAc/EtOH/H2 O 8:2:5:5 (v/v/v/v). After preparation of
to a hybrid LTQ-Orbitrap Discovery XL mass spectrometer (Thermo
the biphasic system, the two phases were separated. The organic
Scientific, Brehmen, Germany), equipped with an electrospray ion-
(upper) phase, containing n-hexane and ethyl acetate, was adjusted
ization (ESI) source. Xcalibur 2.0.7 software (Thermo Scientific) was
to pH 2 by adding TFA (100 mM) and was used as the stationary
used for data acquisition and processing. For all measurements,
phase. The lower aqueous phase comprised the mobile phase, after
the samples were diluted in MeOH/H2 O 80:20 (v/v) at a concen-
adjustment to pH 10 by adding TEA (80 mM). Initially, the column
tration of 200 ␮g/mL. The chromatographic analysis was carried
was filled with the stationary phase at a flow rate of 25 mL/min
out using a Fortis C18 (Fortis Technologies Ltd, Cheshire, UK) col-
and a constant rotation speed of 200 rpm, in descending mode.
umn (100 × 2.1 mm, 1.7 ␮m), heated at 40 ◦ C. The mobile phase
Then, 9.0 g of the extract diluted in 40 mL of acidified upper phase
consisted of 0.1% formic acid in water (solvent A) and acetonitrile
and 5 mL of neutral lower phase were injected. The rotation speed
(solvent B). The gradient program was as follows: initially 5% (B)
was increased to 650 rpm and the pH-zone refining fractionation
for 3 min, then increased until reaching 100% (B) in 18 min, main-
was started by pumping the mobile phase in descending mode. The
tained at 100% (B) for 2 min, and finally decreased to 5% (B) in 2 min
flow rate was set at 10 mL/min for the first 11 fractions, followed
and held at the initial conditions (7 min) for re-equilibration, with
by an increase to 15 mL/min to collect 42 more fractions (elution
a total run time of 32 min. The flow rate was 0.4 mL/min and the
step). The final step of the experiment comprised the extrusion
injection volume was 10 ␮L. MS data acquisition was performed in
of the column content by pumping the organic phase (20 mL/min)
ESI(–) mode in the full scan mass range of m/z 115.0–1000.0, using
in descending mode and the same rotation speed, resulting in
a resolution of 30000. For recording MS2 spectra, data-dependent
the collection of 45 fractions. All collected fractions (98 × 30 mL)
acquisition was selected with a collision-induced dissociation (CID)
were monitored by normal-phase TLC and those presenting similar
value of 35% and a mass resolution of 7500. Capillary temperature
chemical composition were pooled to finally obtain 13 combined
was set at 350 ◦ C and the tuning of capillary voltage and tube lens
fractions (CS-1 to 13), which were concentrated to dryness. Frac-
was at −40 V and −120 V, respectively. Source voltage and source
tions of interest were further analyzed by NMR, LC-HRMS, and
current were set at 2.70 kV and 100 ␮A, respectively. The flow rate
HPLC-PDA to identify the compound structures and estimate the
of N2 was 40 arbitrary units as sheath gas and 10 arbitrary units as
purity of the isolated compounds.
auxiliary gas.

2.6. Thin-layer chromatography (TLC)

2.9. Nuclear magnetic resonance (1 H- & two-dimensional NMR)
TLC analysis was performed on silica gel 60 F254 TLC aluminium spectroscopy
plates of 0.20 mm layer thickness (Merck, Darmstadt, Germany).
Spots were visualized at 254 and 366 nm as well as by spraying NMR spectra were recorded at 300 K on a Bruker Avance III
with a 1:1 mixture of 5% (v/v) H2 SO4 in MeOH and 10% (w/v) vanillin 600 spectrometer (Bruker Biospin GmbH, Rheinstetten, Germany),
in MeOH on the dried plates, followed by heating until maximum operating at 600.11 MHz and equipped with a 5-mm PABBI 1 H/D-
visualization of spots. Solvent systems consisting of DCM/MeOH BB inverse detection probe with a z-gradient. All 1D (1 H) and 2D
98:2 and 95:5 (v/v) were used for chromatogram development. NMR (i.e. COSY, HSQC, HMBC) experiments were carried out by
using default Bruker pulse experiments with standard acquisition
2.7. High-performance liquid chromatography – photodiode parameters, while the solvent used was chloroform (99.8 atom
array detection (HPLC-PDA) % D; Euriso-Top, Saclay, France). The spectra were referenced to
the residual solvent signals at 7.26 and 77.0 ppm for 1 H and 13 C,
The HPLC-PDA analysis of SFE extract, CPC fractions, and puri- respectively. Data acquisition and processing was performed using
fied CBDA was carried out using a SpectraSYSTEM HPLC system TopSpin 2.1 software (Bruker Biospin GmbH).
 J.R. Popp et al. / J. Chromatogr. A 1599 (2019) 196–202 199

Fig. 1. (a) GC–MS chromatograms of the SFE extracts obtained from the fiber-type C. sativa sample, using CO2 and EtOH as co-solvent at different proportions (i.e. 0-5-10-20%),
at a concentration of 1 mg/mL in DCM; (b) comparison of the SFE extracts in terms of the CBD peak area.

3. Results and discussion 3.2. CPC fractionation of extract in pH-zone-refining mode

3.1. Supercritical fluid extraction of C. sativa sample The solvent system selection for performing CPC was carried
out by initially examining the settling time, which was notably
In order to attain purified CBDA, investigation of extraction short (<30 s) in all three cases. For an effective separation using
parameters preceded the isolation procedure. SFE was employed pH-zone-refining methodology, the partition coefficient (K) value
as a green and efficient technology that allows for obtaining clean in the presence of base (Kbase ) must be much lower than 1, while
extracts with high cannabinoid content [43]. In this context, pre- the respective Kacid value should be much greater than 1 [30]. The
liminary experiments were conducted on laboratory scale so as to Kbase value of CBDA using TEA (pH 10) was equal to 0.01, as deter-
evaluate the effect of ethanol as a co-solvent, by examining differ- mined by HPLC-PDA. After adding TFA (pH 2), the Kacid value was
ent proportions up to 20% (w/w). 13.2. It was assumed that TFA is a suitable retainer, since the K value
As revealed by GC–MS analysis, the major compound identified for CBDA in the biphasic solvent system, without pH adjustment,
in all C. sativa extracts was CBD (Table S1). Although CBDA is consid- was 10.6. Moreover, TEA can be an appropriate eluter or displacer
ered the principal cannabinoid present in both hemp plant material for CBDA as well as for other acidic cannabinoids, such as CBDVA,
and unheated extracts thereof, the heating of samples at tempera- given its enhanced lipophilicity as compared with inorganic bases.
tures over 250 ◦ C through this analytical platform leads inevitably Following the selection of the two-solvent system, the CPC frac-
to the decarboxylation of acidic cannabinoids [44]. However, the tionation was performed in pH-zone refining mode resulting in 98
CBD content is to a large degree proportional to the content of CBDA fractions (Table S2). Fractions F1-F8 consisted solely of stationary
and thus could be indicative of the extraction method efficiency. phase, which was eluted until the two phases reached equilibrium
As shown in Fig. 1, the 100% CO2 extract surpassed the extracts inside the column. The volume of the stationary phase eluted was
where EtOH was used as a co-solvent in terms of the CBD peak 240 mL, resulting to a stationary phase retention rate (Sf) equal to
area; the peak area reduction was consistent with the increasing 74%. The Sf value calculated indicates a high number of theoret-
EtOH proportion. ical plates that ensure an efficient separation. Fig. 2 presents the
The chemical composition was almost similar for all extracts CPC chromatogram obtained by monitoring the separation pro-
with respect to the cannabinoids contained, including cannabidiol cedure at 275 nm. The rectangular zone of CBDA was started to
(CBD), cannabidivarin (CBDV), cannabicyclol (CBL), 8 -THC, and form with the increase of pH value. Most of the CBDA contained in
9 -THC (Table S1). Cannabinol (CBN) and cannabielsoin (CBE) are the extract was collected in the fractions F20-F28 (Fig. 2b,c). After
minor cannabinoids that were observed only in the cases of 0% and evaluation of the TLC chromatograms, 13 combined fractions were
5% EtOH proportions. More terpenes were also identified in the generated, namely CS-1 to 13 (Table S2). As shown in Fig. S3, CBDA
extracts obtained using 0% and 5% EtOH, such as ˇ-caryophyllene has been collected in fractions CS-4 to CS-8. Among them, fractions
and humulene epoxide II (Table S1). Taking into account that the CS-5 to CS-7 appeared to be the most promising for the recovery of
SFE extract obtained using 100% CO2 was presumably the most CBDA. Fraction CS-5 comprised F21-F24 (Fig. 2) and accounted for
enriched extract in CBD and accordingly in CBDA, a pilot-scale SFE the 13.2% (w/w) of the complete SFE-CO2 hemp extract (Table 1),
was performed by scaling up the selected methodology. The SFE providing a CBDA purity grade of more than 90% according to the
extraction yield achieved was 8.9% (w/w) and a quantity (9.0 g) of HPLC-PDA analysis. However, this purity related to either salt or
extract was further used for the CPC fractionation. protonated form of CBDA.
200 J.R. Popp et al. / J. Chromatogr. A 1599 (2019) 196–202

Fig. 2. (a) CPC chromatogram of the fractionation using pH-zone-refining methodology, as monitored at 275 nm; (b) fractions F19-F28 collected during elution of CBDVA
and CBDA, and (c) their TLC chromatograms (mobile phase DCM/MeOH 95:5 v/v). Among them, fractions F21-F24 were selected (combined fraction CS-5) to obtain purified
CBDA. The purple dotted line indicates the changes of pH value during the CPC process. (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article).

Table 1
CBDA yield (% w/w) in three combined fractions (CS-5 to CS-7) obtained by CPC, before and after liquid-liquid extraction (LLE).

Fraction Before LLE After LLE

Weight (g) Yielda (% w/w) Weight (g) Yielda (% w/w) Purity (HPLC)

CS-5 1.19 13.2 1.08 12.0 >95%

CS-6 0.48 5.3 0.44 4.9 >90%
CS-7 0.37 4.1 0.34 3.8 >85%
Recovered CBDA content 20.7
Actualb CBDAcontent 25.5
a
Yields were calculated with reference to the amount of SFE-CO2 extract used (9.0 g).
b
As determined by HPLC-PDA.

Thus, CS-5 was submitted to further purification from TEA effective for the isolation of CBDA in terms of purity and cost per-
employing liquid-liquid extraction (LLE) with n-hexane/ethyl formance, compared to previously reported methodologies [25].
acetate 1:1 (v/v) and a water solution of hydrochloric acid at pH 1. No separation of the acidic cannabinoid fraction is required before
The organic (non-polar) fractions collected after 4 replicates of the performing CPC. As it is also well known, the pH-zone-refining
LLE procedure were merged and evaporated to dryness, affording methodology may enhance the CPC column performance, enabling
91% (w/w) recovery of purified CBDA (>95%), based on the HPLC- the use of higher amounts of sample [30].
PDA analysis carried out (Fig. 3). The same procedure was followed Moreover, CPC in descending mode simplifies the isolation of
for the fraction CS-3 (F19), providing 91 mg CBDVA of >85% purity CBDA by reducing the interferences of the lipophilic compounds
grade (HPLC, Fig. S2). The purified CBDA and CBDVA were identi- contained in the SFE-CO2 hemp extract. The separation procedure is
fied by comparison of the NMR data acquired (Figs. S5, S6) with rapid, due to the fact that CBDA starts eluting by pumping a mobile
those previously reported in the literature [45]. LC-HRMS analysis phase volume almost equal to the half column volume (i.e. 0.5 L).
also indicated the CBDA and CBDVA molecular formulas together In particular, more than 65% of CBDA (purity>90%) contained in
with their fragmentation patterns (Figs. S7–S9). The yield in CBDA the extract can be recovered in less than 80 min. No further iso-
(>95%) obtained after CPC and a simple LLE step was estimated to be lation steps that require preparative columns and solid stationary
12.0% (w/w) of the SFE-CO2 hemp extract (Table 1). Similarly, frac- phases are involved. As for the mobile phase, two cost-effective
tions CS-6 and CS-7 accounted for 4.9% and 3.8% of the total extract, solvents of low toxicity are employed, i.e. water and EtOH. Thus,
with a CBDA purity grade of more than 90% and 85%, respectively the proposed CPC method in conjunction with supercritical fluid
(Table 1). extraction constitute a comparatively green approach for the iso-
Considering that the CBDA content of the extract was calculated lation of CBDA and CBDVA. Stability of these acidic cannabinoids
as 25.5% (w/w), this methodology enables a recovery greater than may be also improved as they are mostly collected in Et3 NH+ salt
45% of purified CBDA (>95%, HPLC), more than 65% for CBDA of form. After acidification and LLE of the selected CPC fractions, CBDA
>90% purity and finally, a recovery higher than 80% for CBDA hav- (>95%) and CBDVA (>85%) can be recovered. CBD or CBDV can be
ing a purity >85% (HPLC). The present methodology may be more also derived after decarboxylation through heating.
 J.R. Popp et al. / J. Chromatogr. A 1599 (2019) 196–202 201

Fig. 3. HPLC-PDA chromatograms of isolated CBDA (>95%) and SFE-CO2 C. sativa extract (1 mg/mL); detection at 275 nm.

4. Conclusions [3] B. De Backer, B. Debrus, P. Lebrun, L. Theunis, N. Dubois, L. Decock, A.

Verstraete, P. Hubert, C. Charlier, Innovative development and validation of
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