Enantioselective separation of (±)-β-hydroxy-1,2,3-triazoles by supercritical fluid chromatography and high-performance liquid chromatography

Article abstract of DOI:10.1002/chir.22851

This paper reports the enantioseparation of β-hydroxy-1,2,3-triazole derivatives, which present a broad range of biological properties, by supercritical fluid chromatography (SFC) and high-performance liquid chromatography techniques (HPLC). Polysaccharid

Full text of DOI:10.1002/chir.22851

Received: 11 September 2017
DOI: 10.1002/chir.22851
Revised: 14 February 2018
Accepted: 16 February 2018
R E G U L A R A R T I C L E
Enantioselective separation of ( )‐β‐hydroxy‐1,2,3‐triazoles
by supercritical fluid chromatography and high‐
performance liquid chromatography
Natália Alvarenga
| André L.M. Porto | Juliana Cristina Barreiro
Laboratório de Química Orgânica e
Biocatálise, Instituto de Química de São
Carlos, Universidade de São Paulo, São
Carlos, SP, Brazil
Abstract
This paper reports the enantioseparation of β‐hydroxy‐1,2,3‐triazole derivatives,
which present a broad range of biological properties, by supercritical fluid chro-
matography (SFC) and high‐performance liquid chromatography techniques
(HPLC). Polysaccharide‐based chiral columns (cellulose and amylose) were used
to evaluate the separation in SFC and HPLC. Time of analyses, consumption of
solvent, and parameter optimization were reduced using SFC technique. The col-
umns based on cellulose chiral stationary phase using 2‐propanol and ethanol as
modifiers showed the best results for the enantioresolution of the ( )‐β‐hydroxy‐
1,2,3‐triazoles by SFC analyses. These techniques were applied to evaluate the
selectivity of biocatalytic reduction of β‐keto‐1,2,3‐triazoles by marine‐derived
fungus Penicillium citrinum CBMAI 1186 to obtain the ( )‐β‐hydroxy‐1,2,3‐
triazoles.
Correspondence
Juliana Cristina Barreiro, Laboratório de
Química Orgânica e Biocatálise, Instituto
de Química de São Carlos, Universidade
de São Paulo, Av. João Dagnone, 1100 Ed.
Química Ambiental, J. Santa Angelina,
13563‐120, São Carlos, SP, Brazil.
Email: juliana.barreiro@gmail.com
Funding information
Conselho Nacional de Desenvolvimento
Científico e Tecnológico ‐ CNPq, Grant/
Award Number: 141844/2013‐2;
Coordenação de Aperfeiçoamento de
Pessoal de Nível Superior; CAPES/PNPD,
São Carlos Institute of Chemistry, University
of São Paulo
KEYWORDS
biocatalysis, enantiomeric separation, HPLC, SFC, triazoles
1 | INTRODUCTION
enantioresolution of chiral compounds, mainly in the phar-
maceutical industry for purification and enantioseparation
High‐performance liquid chromatography (HPLC) is the
major separation technique used for the qualitative and
quantitative analysis of chiral compounds.1,2 The
widespread use of HPLC may be attributed to advances
on the development of chiral stationary phases (CSPs),
which improved the separation of racemic compounds.3,4
Nevertheless, several limitations may be disadvantageous
to the development of chiral methods by HPLC, such as
long equi‐libration time and long analysis time and the
use of toxic and flammable solvents. Another drawback
for enantiomeric purity determination is the effect of peak
broadening caused by diffusion processes in HPLC, which
can affect the quality of the separation contributing to
low efficiencies.3,5
of drugs and their impurities, improving the resolution and
reducing analysis time.6 SFC is considered as an environ-
mental friendly technique due to reduced consumption of
toxic solvents and additives.7,8 Carbon dioxide and polar
modifiers (e.g., alcohols and acetonitrile) are used as mobile
phases coupled with a wide variety of stationary phases.
The high diffusivity, low viscosity, and solvating power
afforded by the use of CO2 in the mobile phase is responsible
for the success of SFC on the chiral separations.8,9
The polysaccharides‐based derivatives (e.g., cellulose
and amylose) CSPs are extensively used for HPLC as well
as SFC enantioseparations due to versatility, durability,
and sample loadability.1,10 Comparative studies of
enantioselective separations have been showed that both
SFC and HPLC provide good selectivity; however, the
advantage of SFC over HPLC occurs in terms of flow rate,
Supercritical fluid chromatography (SFC) has emerged as
an alternative technique to HPLC for routine applications in
Chirality. 2018;1–10.
wileyonlinelibrary.com/journal/chir
© 2018 Wiley Periodicals, Inc.
1

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ALVARENGA ET AL.
FIGURE 1 ( )‐β‐Hydroxy‐1,2,3‐triazole derivatives evaluated at
the study of enantioselective separation by SFC and HPLC
resolution, time of analysis, consumption of solvent, and
high throughput.1,8,9,11 Thus, the SFC has been used not
only for purification purpose, but also for method
development and screening of chiral and achiral
compounds in different matrices.12,13
SCHEME 1 Synthesis of 2‐azido‐1‐phenylethanone derivatives
with the respective percentage of yield
Therefore, in this work these two techniques were
employed for the chiral separation of β‐hydroxy‐1,2,3‐
triazoles,14 which are an important class of heterocyclic
compounds obtained only by synthetic methodologies.
These compounds have been extensively studied as poten-
tial targets for drug discovery since they possess a broad
range of biological properties, including antimicrobial,
antiviral, antiepileptic, anti‐HIV and activities against
several neglected diseases.15
(Barcelona, Spain). All reagents and solvents were used
without further purification. Salts used in the preparation
of artificial seawater were purchased from Synth, Merck,
or Vetec (Brazil). Malt extract was purchased from
Acumedia (Indaituba, Brazil), and Agar was purchased
from Himedia (Mumbai, India).
The racemic standards of β‐hydroxy‐1,2,3‐triazole
were prepared as described below by the following steps
(Schemes 1, 2, and 3).
Racemic β‐hydroxy‐1,2,3‐triazole derivatives (Figure 1)
were obtained through the reduction of β‐keto‐1,2,3‐
triazoles using NaBH4. The racemic compounds were used
as analytical standard to evaluate the enantioselective
bioreduction of β‐keto‐1,2,3‐triazoles by marine‐derived
fungus16 Penicillium citrinum CBMAI 1186.
CSP‐base enantioselective separation has been an
extremely useful tool in biocatalytic studies.17 The
asymmetric synthesis of chiral alcohols by biocatalysis
2.2 | Synthesis of 2‐azido‐1‐
phenylethanone derivatives
The 2‐azido‐1‐phenylethanones were synthetized by reac-
tion of 2‐bromo‐1‐phenylethanone derivatives (5.00 mmol)
and sodium azide (10.0 mmol) in round‐bottomed flask
(100 mL) containing acetone (30 mL), followed by stirring
at room temperature for 4 to 6 hours (Scheme 1). The
reactions were monitored by TLC and extracted with
EtOAc (3 × 20.0 mL). The combined organic layers were
dried over anhydrous Na2SO4, filtered, and evaporated
under reduced pressure. The products were purified by
column chromatography on silica gel and eluted with
mixtures of hexane and EtOAc (7:3). The 2‐azido‐1‐
phenylethanones were characterized by spectroscopic
consists in
a well‐known methodology to obtain
enantiomerically pure compounds. Thus, based on the
HPLC method previously established to monitor the
bioreduction of β‐keto‐1,2,3‐triazole by P. citrinum
CBMAI 1186, we evaluated the potential of SFC tech-
nique on the enantioselective separation for these
compounds.
1
analyses H NMR, 13C NMR, and IR.16
2 | MATERIALS AND METHODS
2.1 | Chemical and reagents
2.3 | Synthesis of β‐keto‐1,2,3‐triazoles
The β‐keto‐1,2,3‐triazoles were synthetized by the
reaction of 2‐azido‐1‐phenylethanones derivatives
(1.24 mmol), phenylacetylene (1.86 mmol), (+)‐sodium
L‐ascorbate (10% mol), and CuSO4.5H2O (1% mol) in
round‐bottomed flask (50.0 mL) containing distilled water
(20.0 mL), followed by stirring at room temperature for
4 hours (Scheme 2). The reactions were monitored by
TLC and extracted with EtOAc (3 × 20.0 mL). The
combined organic layers were dried over anhydrous
Na2SO4, filtered, and evaporated under reduced pressure.
The products were purified by column chromatography
Sodium borohydride (97%) was purchased from Vetec
(Duque de Caxias, Brazil) and sodium azide from Merck
(São Paulo, Brazil). The 2‐bromo‐1‐phenylethanone 98%,
2‐bromo‐1‐(4‐chlorophenyl)ethanone 98%, 2‐bromo‐1‐(4‐
bromophenyl)ethanone 98%, phenylacetylene 98%, and
(+)‐sodium L‐ascorbate 98% were purchased from
Sigma‐Aldrich (São Paulo, Brazil). Ethyl acetate (EtOAc),
hexane, and acetone were purchased from Synth (São
Paulo, Brazil). Hexane, 2‐propanol (IPA), acetonitrile
(ACN), and ethanol (EtOH) were purchased from Panreac

ALVARENGA ET AL.
3
SCHEME 2 Synthesis of β‐keto‐1,2,3‐
triazoles with the respective percentage
of yield
on silica gel and eluted with mixtures of hexane and
medium composition and fungus cultivation were
described elsewhere.18
EtOAc (2:1). The β‐keto‐1,2,3‐triazoles were characterized
1
by spectroscopic analyses H NMR, 13C NMR, and IR.16
2.6 | Bioreduction of ( )‐β‐keto‐1,2,3‐
triazoles by the marine‐derived fungus
P. citrinum CBMAI 1186
2.4 | Synthesis of ( )‐β‐hydroxy‐1,2,3‐
triazoles
The ( )‐β‐hydroxy‐1,2,3‐triazoles 1 to 6 were synthetized
by the addition of β‐keto‐1,2,3‐ketotriazoles (50 mg) in
round‐bottomed flask (50 mL) containing distilled
methanol (20 mL) and sodium borohydride (1.5
equivalent) on ice bath (T = 0°C), followed by stirring
for 1.5 hours (Scheme 3). The reactions were monitored
by TLC, and the solvent was evaporated under pressure
and the residue extracted with EtOAc (3 × 10.0 mL).
The combined organic layers were dried over anhydrous
Na2SO4, filtered, and evaporated under reduced pressure.
The products were purified by column chromatography
on silica gel and eluted with mixtures of dichloromethane
hexane and methanol (85:15:5). The ( )‐β‐hydroxy‐1,2,3‐
triazoles 1 to 6 were characterized by spectroscopic
The biocatalytic reductions were performed with 2 g of
wet mycelium from P. citrinum CBMAI 1186 and ( )‐β‐
keto‐1,2,3‐triazoles (20 mg) dissolved in dimethylsulfoxide
(300 μL), in 95 mL of phosphate buffer solution
(Na2HPO4/KH2PO4, pH 5, 0.07 M) sterilized in autoclave
(121°C, 1.5 kPa) and 5 mL of methanol (5% v/v) sterilized
in ultraviolet light (20 min). The mixtures were incubated
in orbital shaker for 12 days (32°C, 130 rpm). After 12 days
of incubation, the reaction was filtered in a Buchner
funnel and the mycelial mass obtained was transferred
to a 250‐mL Erlenmeyer flask and suspended in 60 mL
of distilled water and EtOAc (1:1). This biphasic mixture
was submitted to magnetic stirring for 30 min, filtered
again together with the medium obtained in the previous
filtration and extracted with EtOAc (3 × 25.0 mL). The
combined organic layers were dried over anhydrous
Na2SO4, filtered, and evaporated under reduced pressure.
The products were purified by column chromatography
on silica gel and eluted with a mixture of dichlorometh-
ane, hexane, and methanol (85:15:5). The aliquot of puri-
fied sample was suspended in 1.5 mL of 2‐propanol or
methanol.
1
analyses H NMR, 13C NMR, and IR.16
2.5 | Marine‐derived fungus Penicillium
citrinum CBMAI 1186
The isolation of marine‐derived fungus P. citrinum
CBMAI 1186 was previously described in Ferreira
et al.18 The procedures for the preparation of culture
SCHEME 3 Synthesis of ( )‐β‐hydroxy‐
1,2,3‐triazoles with the respective
percentage of yield

4
ALVARENGA ET AL.
5.0 μm, 0.46 cm × 250 mm) for ( )‐β‐hydroxy‐1,2,3‐
triazoles 2 to 6. The mobile phase was hexane:2‐propanol
(v:v) with flow rate of 0.5 mL min−1 (( )‐β‐hydroxy‐1,2,3‐
triazole 1–5) and 0.6 mL min−1 (( )‐β‐hydroxy‐1,2,3‐tri-
azole 6), oven temperature at 40°C and injection volume
of 10 μL. All the solutions were prepared using 1 mg of
each compound dissolved in methanol or 2‐propanol.
3 | INSTRUMENTATION
SFC
A Waters ACQUITY UPC2 system, equipped with an
ACQUITY UPC2 binary solvent manager, ACQUITY
UPC2 sample manager, Automatic Back‐Pressure
Regulator (ABPR), and an ACQUITY photodiode array
(PDA) detector was used to perform the SFC experiments.
Data acquisition was performed using the Waters
EmpowerTM 3 software. The injector was equipped with a
10 μL loop. Peaks were registered at a wavelength of 190
to 800 nm using compensated mode (380‐480 nm). Three
columns were used to evaluate the separation of the ( )‐β‐
hydroxy‐1,2,3‐triazole derivatives: ACQUITY UPC2 Trefoil
AMY 1 (Amylose tris‐[3,5‐dimethylphenylcarbamate]–
2.5 μm, 3.0 mm × 150 mm), ACQUITY UPC2 Trefoil CEL
1 (Cellulose tris‐[3,5‐dimethylphenylcarbamate]–2.5 μm,
3.0 mm × 150 mm), and ACQUITY UPC2 Trefoil CEL 2
3.1 | Data analysis
The separation parameters such as retention factor (k)
(Equation 1), selectivity (or separation factor) (α)
(Equation 2) and resolution (Rs) (Equation 3) were
calculated using the following equations6,19
:
tR−t0
k' ¼
;
(1)
(2)
(3)
t0
k2
k1
a ¼
;
(Cellulose
tris‐[3‐chloro‐4‐methylphenylcarbamate]–
2.5 μm, 3.0 mm × 150 mm). EtOH, IPA, and ACN were
used as the co‐solvent. The SFC binary pump was set up
to initial pump with 20% of co‐solvent at a flow rate of
1.0 ml min−1. The gradient elution was linear from 20%
to 60% of co‐solvent in 6 minutes and then from 60% to
0% of co‐solvent in 2 minutes. The density of the
subcritical fluid was regulated via column temperature
of 35°C and automated backpressure regulator (ABPR)
setting of 13.8 (MPa), with injection volume of 2 μL.
t2−t1
RS ¼ 2
:
w1 þ w2
4 | RESULTS AND DISCUSSION
4.1 | Enantiomeric separation of ( )‐β‐
hydroxy‐1,2,3‐triazoles by HPLC
The polysaccharide‐based CSPs columns have been widely
used for screening of molecules under different mobile
phase conditions. In general, the mechanisms evolved on
the enantiodiscrimination of the enantiomers by polysac-
charide‐based CSPs are based on the spatial effects of
cavities built by the polysaccharide, steric effects, π‐π
interactions, hydrogen bonding and dipole‐dipole.20,21
Two polysaccharide‐based CSPs, OD‐H (cellulose
tris(3,5‐dimethylphenylcarbamate) and AS‐H (amylose
tris[(S)‐α‐methylbenzylcarbamate]) were used to improve
the separation of ( )‐β‐hydroxy‐1,2,3‐triazole derivatives
by HPLC. Table 1 shows the results of retention factor,
HPLC
The HPLC analyses were performed on a Shimadzu Prom-
inence equipped with a LC‐20AT pump, a SIL‐20AHT
injector system with a 100‐μL loop, and SPD‐M20A diode
array detector with a CBM‐20A interface. Data acquisition
was performed using the LC Solution software. The chiral
columns used were CHIRACEL OD‐H (cellulose tris [3,5‐
dimethylphenylcarbamate]–5.0 μm, 0.46 cm × 250 mm)
for ( )‐β‐hydroxy‐1,2,3‐triazole
AS‐H (amylose tris [(S)‐α‐methylbenzylcarbamate]–
1 and CHIRALPAK
TABLE 1 Retention factor (k), selectivity (α), and resolution (Rs) for the ( )‐β‐hydroxy‐1,2,3‐triazoles 1‐6 by HPLC analyses
Compounds
tR, min
Mobile Phase (v:v)
k1
k2
α
Rs
1
2
3
4
5
6
65
35
57
65
60
60
85:15
75:25
90:10
90:10
90:10
80:20
8.22
3.15
6.52
7.21
6.02
3.90
8.78
3.80
7.18
8.03
6.66
6.74
1.07
1.21
1.10
1.13
1.11
1.75
0.61
1.85
1.06
1.18
0.91
4.33
ta = analysis time; k1 = retention factor for the peak 1 racemic compound; k2 = retention factor for the peak 2 of the racemic compound. CHIRACEL OD‐H and
CHIRALPAK AS‐H were used on the enantioseparation of compounds 1 and 2‐6, respectively. Hexane:2‐propanol (v:v) was used as mobile phase at different
compositions. A flow rate of 0.5 mL min−1 for the compounds 1‐5 and 0.6 mL min−1 for the 6.

ALVARENGA ET AL.
5
selectivity and resolution previously established for ( )‐β‐
hydroxy‐1,2,3‐triazoles 1 to 6.
(acetonitrile, 2‐propanol or ethanol). The retention time
for all compounds was decreased when SFC was used.
From the results shown on Table 2 for SFC analyses, it
can be observed that changes on the nature of co‐solvent
has different effects on CSPs columns. The improvement
on the enantioselectivity using polysaccharide‐based CSPs
and co‐solvent with different proton‐acceptor characters
by SFC was brightening discussed by Khater and West.22
Based in a series of chemometric studies considering the
type of based‐CSPs and nature of co‐solvent, the authors22
highlighted that when the percentage of modifier is high
(above 15%‐20%) the elution can be more affected by
interactions between analyte‐mobile phases than the ana-
lyte‐stationary phases. Better selectivity and resolution
were achieved for all compounds (1 to 6) on cellulose
derivatives based‐columns CEL 1 and CEL 2 using 2‐
propanol or ethanol as co‐solvent in comparison with
AMY 1. The selectivity of the compounds with lower pro-
ton‐donor capability demonstrated less difference in the
presence of co‐solvents (EtOH and IPA) when compared
to the compounds with proton‐donor character (3 to 6).
On both, the enantioseparation was driven by hydrogen
interactions.
The determination of the exact mechanisms behind
the enantiomeric separation is a difficult task. π‐π
Interactions between the phenyl moieties of ( )‐β‐
hydroxy‐1,2,3‐triazole derivatives and the aromatic ring
of the chiral selector from AS‐H and OD‐H CSP can be
evolved on the chiral discrimination of the enantiomers.
Partial separation (RS = 0.61‐1.18) was observed for
the compound 1 (85:15, v:v) and compounds 3, 4, and 5
(90:10, v:v) on CSP OD‐H and AS‐H, respectively, and
complete resolution was achieved for the compounds 2
and 6 (Rs > 1.5). Besides the π‐π interactions, the groups
R═OCH3 and NO2, present in the compounds 2 and 6,
respectively, a hydrogen bond may be formed with ─NH
group at AS–H chiral column. Moreover, the groups
OCH3 and NO2 can interact by dipole‐dipole bond with
the C═O present at the AS–H CSP contributing to
increase the selectivity for these compounds.19
4.2 | Enantiomeric separation of ( )‐β‐
hydroxy‐1,2,3‐triazoles by SFC
The Figure 2 presents the chromatogram for the enan-
tiomeric separation of ( )‐β‐hydroxy‐1,2,3‐triazole 2 at
CEL 1 using different co‐solvents, illustrating the better
separation in presence of EtOH (Rs = 6.77, α = 1.32).
The retention factors in SFC were smaller than in
HPLC for all compounds. This is an indicative that the
Polysaccharide‐based CSPs used on the SFC analyses
were amylose derivative (AMY 1) and cellulose
derivatives (CEL 1 and CEL 2). Table 2 shows the results
obtained on the enantioseparation of β‐hydroxy‐1,2,3‐tri-
azole derivatives 1 to 6 by SFC. The eluent used in these
columns were mixtures of carbon dioxide‐co‐solvent
TABLE 2 Retention factor (k), selectivity (α), and resolution (Rs) for the ( )‐β‐hydroxy‐1,2,3‐triazoles by SFC analyses
Column
Compounds
1
Mobile Phase (v:v)
k1
1.92
k2
1.99
α
Rs
ACN
IPA
1.03
1.09
1.23
0.95
0.69
0.95
1.66
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
0.83
0.94
n.d.
1.09
0.64
2.09
1.39
0.83
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
1.74
0.96
n.d.
1.16
0.68
2.22
1.45
0.92
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
1.78
1.00
n.d.
1.06
1.06
1.06
1.05
1.11
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
1.03
1.04
n.d.
EtOH
ACN
IPA
2
3
4
5
EtOH
ACN
IPA
AMY 1
EtOH
ACN
IPA
EtOH
ACN
IPA
EtOH
(Continues)

6
ALVARENGA ET AL.
TABLE 2 (Continued)
Column
Compounds
Mobile Phase (v:v)
ACN
IPA
k1
1.31
k2
1.35
α
Rs
6
1
2
3
4
5
6
1
2
3
4
5
6
1.03
1.25
2.18
1.83
n.d.
6.95
7.42
0.67
4.57
8.41
n.d.
3.53
8.10
n.d.
3.37
6.93
n.d.
3.86
6.10
1.30
1.03
6.77
…
1.46
0.95
n.d.
0.84
1.27
2.66
0.95
1.34
n.d.
0.97
1.49
n.d.
1.14
1.78
n.d.
0.62
0.93
2.44
1.17
1.54
…
1.60
1.05
n.d.
1.13
1.76
2.80
1.21
1.89
n.d.
1.14
2.02
n.d.
1.31
2.32
n.d.
0.79
1.33
2.74
1.22
2.04
…
1.10
1.10
n.d.
1.35
1.38
1.05
1.28
1.41
n.d.
1.17
1.36
n.d.
1.15
1.31
n.d.
1.26
1.43
1.13
1.05
1.32
…
EtOH
ACN
IPA
EtOH
ACN
IPA
EtOH
ACN
IPA
CEL 1
EtOH
ACN
IPA
EtOH
ACN
IPA
EtOH
ACN
IPA
EtOH
ACN
IPA
0.74
0.50
n.d.
1.01
0.74
n.d.
0.78
0.55
n.d.
0.95
0.71
n.d.
0.50
0.29
n.d.
0.99
0.73
0.97
0.66
n.d.
1.20
0.88
n.d.
1.04
0.72
n.d.
1.23
0.90
n.d.
0.70
0.42
n.d.
1.16
0.89
1.31
1.32
n.d.
1.19
1.19
n.d.
1.33
1.31
n.d.
1.30
1.27
n.d.
1.40
1.45
n.d.
1.17
1.22
3.90
3.03
n.d.
2.39
2.49
n.d.
3.59
3.14
n.d.
4.31
3.22
n.d.
3.74
2.92
n.d.
2.50
2.78
EtOH
ACN
IPA
EtOH
ACN
IPA
CEL 2
EtOH
ACN
IPA
EtOH
ACN
IPA
EtOH
ACN
IPA
EtOH
k1 = retention factor for the peak 1 of racemic compound; k2 = retention factor for the peak 2 of the racemic compound. SFC conditions: Gradient
elution was linear from 20% to 60% of co‐solvent in 6 min and then from 60% to 0% of co‐solvent in 2 min. Column temperature = 35°C and ABPR setting
of 13.8 (MPa).

ALVARENGA ET AL.
7
2.20
2.00
1.80
1.60
1.40
1.20
1.00
0.80
0.60
0.40
0.20
0.00
(A)
(B)
3.00
2.50
2.00
1.50
1.00
0.50
0.00
(C) 3.00
2.50
2.00
1.50
1.00
0.50
0.00
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00
Time (min)
FIGURE 2 Chromatograms of ( )‐β‐hydroxy‐1,2,3‐triazole 2 obtained at column CEL 1 in different mobile phases. A, ACN, B, IPA, and C,
EtOH
(A)1.40
1.20
1.00
0.80
0.60
0.40
0.20
0.00
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00
Time (min)
mAU
(B)
245nm4nm (1.00)
125
100
75
50
25
0
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
45.0
50.0
55.0
60.0
min
Time (min)
FIGURE 3 Chromatograms of ( )‐β‐hydroxy‐1,2,3‐triazole 1 obtained by A, SFC at column CEL 2 using EtOH as modifier and B, HPLC at
column OD‐H using hexane:2‐propanol (85:15) as mobile phase, λ = 245 nm

8
ALVARENGA ET AL.
(A)2.00
1.80
1.60
1.40
1.20
1.00
0.80
0.60
0.40
0.20
0.00
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00
Time (min)
mAU
245nm4nm (1.00)
(B)
200
175
150
125
100
75
50
25
0
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
45.0
50.0
55.0
min
Time (min)
FIGURE 4 Chromatograms of ( )‐β‐hydroxy‐1,2,3‐triazole 5 obtained by A, SFC at column CEL 2 using EtOH as modifier and B, HPLC at
column AS‐H using hexane:2‐propanol (90:10) as mobile phase, λ = 245 nm
(A)
1.20
1.00
0.80
0.60
0.40
0.20
0.00
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00
Time (min)
mAU
251nm,4nm (1.00)
(B) 250
225
200
175
150
125
100
75
50
25
0
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
45.0
50.0
55.0
min
Time (min)
FIGURE 5 Chromatograms of ( )‐β‐hydroxy‐1,2,3‐triazole 6 obtained by A, SFC at column CEL 2 using EtOH as modifier and B, HPLC at
column AS‐H using hexane:2‐propanol (80:20) as mobile phase, λ = 251 nm
elution strength of the carbon dioxide‐IPA and carbon
dioxide‐EtOH is higher than the hexane:IPA (v:v).
These facts emphasize that the solute‐mobile phase and
mobile phase‐stationary phase are not equivalent for
both techniques.2 Thereby, the separation of compound 1
was achieved in the SFC analysis, with good RS, using
CEL 1 column.
No differences were observed by the use of modifier
IPA or EtOH on the CEL 2 columns for the selectivity
and resolution of the enantiomers.

ALVARENGA ET AL.
9
(A)
(B)
FIGURE 6 Chromatograms of ( )‐β‐hydroxy‐1,2,3‐triazole 6 and (S)‐(+)‐β‐hydroxy‐1,2,3‐triazole 6 obtained by biotransformation with
P. citrinum CBMAI 1186 by A, SFC at column CEL 2 using EtOH as modifier and B, HPLC at column AS‐H using hexane:2‐propanol (80:20)
as mobile phase, λ = 251 nm
Figures 3–5 present the chromatograms obtained for
the ( )‐β‐hydroxy‐1,2,3‐triazoles 1, 5, and 6 by SFC and
HPLC.
Figure 6 shows the chromatograms of the standard
racemic mixture and the product of biotransformation
by P. citrinum CBMAI 1186 using both techniques, HPLC
and SFC.
ORCID
Natália Alvarenga http://orcid.org/0000-0003-0751-2685
Juliana Cristina Barreiro http://orcid.org/0000-0002-3657-
2620
REFERENCES AND NOTES
The biotransformation reaction for the by P. citrinum
CBMAI 1186 presented high selectivity towards the
formation of (S)‐(+)‐β‐hydroxy‐1,2,3‐triazole 6 with 96%
of enantiomeric excess. It is noteworthy that the elution
order for (S)‐(+)‐β‐hydroxy‐1,2,3‐triazole 6 was inverted
for the AS–H in HPLC and the CEL 2 for SFC.
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ACKNOWLEDGMENTS
The authors acknowledge financial support from the
Brazilian agencies CAPES (J.C. Barreiro–CAPES/PNPD,
São Carlos Institute of Chemistry, University of São
Paulo) and CNPq (MSc N. Alvarenga–CNPq 141844/
2013‐2). Also, thanks to Prof Dr M. W. Paixão (Federal
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How to cite this article: Alvarenga N, Porto
ALM, Barreiro JC. Enantioselective separation of
( )‐β‐hydroxy‐1,2,3‐triazoles by supercritical fluid
chromatography and high‐performance liquid
chromatography. Chirality. 2018;1–10. https://doi.
org/10.1002/chir.22851
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phenylethanone derivatives by whole cells of marine‐derived
fungi applied to synthesis of enantioenriched β‐hydroxy‐1,2,3‐
triazoles. Biocatal Biotransformation. 2017;35:1–9.
17. Joyce LA, Regalado EL, Welch CJ. Hydroxypyridyl imines:
enhancing chromatographic separation and Stereochemical