Reliable HPLC separation, vibrational circular dichroism spectra, and absolute configurations of isoborneol enantiomers

Article abstract of DOI:10.1002/chir.22728

Resolution of chiral compounds has played an important role in the pharmaceutical field, involving detailed studies of pharmacokinetics, physiological, toxicological, and metabolic activities of enantiomers. Herein, a reliable method by high-performance liquid chromatography (HPLC) coupled with an optical rotation detector was developed to separate isoborneol enantiomers. A cellulose tris(3, 5-dimethylphenylcarbamate)-coated chiral stationary phase showed the best separation performance for isoborneol enantiomers in the normal phase among four polysaccharide chiral packings. The effects of alcoholic modifiers and column temperature were studied in detail. Resolution of the isoborneol racemate displayed a downward trend along with an increase in the content of ethanol and column temperature, indicating that less ethanol in the mobile phase and lower temperature were favorable to this process. Moreover, two isoborneol enantiomers were obtained via a semipreparative chiral HPLC technique under optimum conditions, and further characterized by analytical HPLC, and experimental and calculated vibrational circular dichroism (VCD) spectroscopy, respectively. The solution VCD spectrum of the first-eluted component was consistent with the Density Functional Theory (DFT) calculated pattern based on the SSS configuration, indicating that this enantiomer should be (1S, 2S, 4S)-(+)-isoborneol. Briefly, these results have provided reliable information to establish a method for analysis, preparative separation, and absolute configuration of chiral compounds without typical chromophoric groups.

Full text of DOI:10.1002/chir.22728

Received: 21 December 2016
DOI: 10.1002/chir.22728
Revised: 11 May 2017
Accepted: 15 May 2017
R E G U L A R A R T I C L E
Reliable HPLC separation, vibrational circular dichroism spectra,
and absolute configurations of isoborneol enantiomers
Rui‐Qi Gao1 | Jun Fan1
| Qi Tan1 | Dong Guo2 | Tao Chen1 | Ru‐Jian He1 | Dan Li3 |
Hui Zhang3 | Wei‐Guang Zhang1
1 School of Chemistry and Environment,
South China Normal University, Guangzhou,
China
2 Guangdong YanJie Pharmatech Co. Ltd,
Guangzhou, China
3 School of Chemistry and Chemical
Engineering, Xiamen University, Xiamen,
China
Abstract
Resolution of chiral compounds has played an important role in the pharmaceu-
tical field, involving detailed studies of pharmacokinetics, physiological, toxico-
logical, and metabolic activities of enantiomers. Herein, a reliable method by
high‐performance liquid chromatography (HPLC) coupled with an optical rota-
tion detector was developed to separate isoborneol enantiomers. A cellulose
tris(3, 5‐dimethylphenylcarbamate)‐coated chiral stationary phase showed the best
separation performance for isoborneol enantiomers in the normal phase among
four polysaccharide chiral packings. The effects of alcoholic modifiers and col-
umn temperature were studied in detail. Resolution of the isoborneol racemate
displayed a downward trend along with an increase in the content of ethanol
and column temperature, indicating that less ethanol in the mobile phase and
lower temperature were favorable to this process. Moreover, two isoborneol
enantiomers were obtained via a semipreparative chiral HPLC technique under
optimum conditions, and further characterized by analytical HPLC, and experi-
mental and calculated vibrational circular dichroism (VCD) spectroscopy, respec-
tively. The solution VCD spectrum of the first‐eluted component was consistent
with the Density Functional Theory (DFT) calculated pattern based on the SSS
configuration, indicating that this enantiomer should be (1S, 2S, 4S)‐(+)‐
isoborneol. Briefly, these results have provided reliable information to establish
a method for analysis, preparative separation, and absolute configuration of chiral
compounds without typical chromophoric groups.
Correspondence
J. Fan or W.‐G. Zhang, School of Chemistry
and Environment, South China Normal
University, Guangzhou, 510006, China.
Email: fanj@scnu.edu.cn; wgzhang@scnu.
edu.cn
Funding information
National Natural Science Foundation of
China, Grant/Award Number:
NSFC‐21571070, NSFC‐21171059,
NSFC‐21603076 and NSFC‐21273175;
Guangdong Provincial Science and
Technology Project, Grant/Award Number:
GPSTP‐2016B090921005, GPSTP‐
2016B010108007 and GPSTP‐
2014A010101145; Guangzhou Science and
Technology Project, Grant/Award Number:
GSTP‐201508020093 and GSTP‐
201604020145
KEYWORDS
absolute configuration, HPLC enantioseparation, isoborneol, optical rotation detector, vibrational circular
dichroism
1 | INTRODUCTION
chemicals, etc. High‐performance liquid chromatography
(HPLC) with various chiral stationary phases (CSPs) has
Enantiomers of chiral compounds, especially chiral pharma-
ceuticals, have shown significant differences in pharmacol-
ogy activities, metabolic processes, and toxicities in human
bodies.1-3 Enantioseparation methods are the subjects of
growing interest in the life sciences, pharmaceuticals, fine
been one of most commonly used techniques for chiral
analysis, determination, and preparative separation of chiral
compounds over the past decades.4-6
Monoterpenoid compounds including hydrocarbon
monoterpenes and oxygenated monoterpenes are very often
Chirality. 2017;1–8.
wileyonlinelibrary.com/journal/chir
© 2017 Wiley Periodicals, Inc.
1

2
GAO ET AL.
found in plants and have been widely used as flavors,
fragrances, pharmacological substances, etc. Enantiomers
show significant differences in sensory qualities, odor
threshold, pharmacology activities, metabolic processes,
and toxicities.7-9 Since chiral monoterpene compounds
possess few or no functional groups and high volatility, their
chiral analysis studies have been mainly performed by gas
chromatography with cyclodextrin derivatives as chiral selec-
tors.7,10-12 In order to obtain optically active stereoisomers in
batch, several research groups tried to develop direct HPLC
separation methods with chiral columns. In Zukowski's work,
complete separation of ( )‐pinene with a selectivity factor of
1.53 was obtained by HPLC with α‐cyclodextrin as the
mobile phase additive.13 Armstrong and Zukowski investi-
gated enantioseparation of three hydrocarbon monoterpenes,
namely, ( )‐pinene, ( )‐β‐pinene, and ( )‐camphene, on a
native α‐cyclodextrin bonded phase through HPLC with a
methanol–water mixture, and the effect of analyte concentra-
tion as well as mobile phase additives on selectivity and
efficiency were also discussed.14 In 2002, HPLC chiral
analyses of monoterpenoids spread to the oxygenated deriva-
tives. Tamura's group reported the HPLC separation of nine
chiral monoterpenes on amylose, cellulose, and β‐cyclodex-
trin phenylcarbamate chiral stationary phases in the
reversed‐phase mode.15 The amylose column showed a good
chiral recognition performance for seven analytes, especially
for linalool, and the configuration of linalool was directly
determined with this chiral HPLC system coupled with an
optical rotatory (OR) detector. However, the cellulose
column showed a very poor ability in chiral recognition of
these analytes. One more thing should be noted, that most
of their retention time was very long on the amylose column
and the separation work seemed to be time‐consuming. Till
now, the studies concerned with the resolution of monoter-
penes by chiral HPLC have been rarely reported.
of ischemia‐induced myocardial damage, treatment of central
nervous system diseases, stimulation of neurogliocyte divi-
sion and growth, etc.16-19 In addition, the OR and refractive
index (RI) detectors became the preferred choices in HPLC
studies for the analytes with nonultraviolet active groups.
Especially the OR detector as a highly selective detector is
totally free from nonoptically active compounds.20-22
Goodall's group investigated the semipreparation of warfarin
enantiomers through HPLC with an improved OR detector.21
Our group reported a comparative evaluation of HPLC
coupled with UV, OR, RI, and evaporative light scattering
detectors for qualitative and quantitative analyses of
metalaxyl racemate, and the effects of separation conditions
on the peak area ratio between two enantiomers, including
sample concentration, column temperature, mobile phase
composition, and flow rate, have been investigated in
detail.22 In this work, isoborneol enantiomers were success-
fully resolved on the cellulose derivative‐coated CSP by
HPLC with a hexane‐ethanol mixture. Taking its resolution
into consideration, the effects of types of CSPs, alcoholic
modifiers, and column temperature are discussed in detail.
Under optimal conditions, chiral separation of isoborneol
was done within 10 min. Moreover, two optically pure
isoborneol enantiomers were obtained by semipreparative
HPLC with the OR detector, and their absolute configura-
tions (AC) were determined by comparison with the experi-
mental and calculated vibrational circular dichroism (VCD)
spectra.
2 | MATERIALS AND METHODS
2.1 | Chemicals and materials
Racemic isoborneol (purity 90.0%, GC) was bought from TCI
Chemicals (Shanghai, China). All HPLC‐grade solvents were
purchased from LabScience (Pittsburgh, PA). Four analytical
columns (Enantiopak OD, OX, OJ, and AD, 250 × 4.6 mm,
5 μm) and one semipreparative Enantiopak OD chiral column
(250 × 10 mm, 5 μm) were kindly provided by Guangzhou
Research & Creativity Biotechnology (Guangzhou, China).
The Enantiopak OD chiral columns were packed with cellu-
lose tris(3, 5‐dimethylphenylcarbam ate)‐coated silica gel,
the Enantiopak OJ column was packed with cellulose tris
(4‐methylbenzoate)‐coated silica gel, the Enantiopak OX col-
umn was packed with cellulose tris(4‐cholor‐3‐methylphenyl
carbamate)‐coated silica gel, and the Enantiopak AD column
was packed with amylose tris (3, 5‐dimethylphenylcarbamate)‐
coated silica gel.
Herein, we focused on the enantioseparation of
isoborneol (Scheme 1) through HPLC with polysaccharide
chiral columns and an OR detector. As an epimeride of
borneol, isoborneol shows good biological activities in
antiinflammation, antibacterial, analgesia, and as an antivi-
rus, and has been applied in contribution to pharmaceutical
absorption, treatment of coronary artery spasm, abatement
2.2 | Instruments and chromatography
Enantioseparation of isoborneol was run with an Agilent
(Palo Alto, CA) 1200 series HPLC system equipped with a
SCHEME 1 Molecular structures of a pair of isoborneol enantiomers

GAO ET AL.
3
vacuum degasser, a quaternary pump, an auto injector with a
100 μL sample loop, a column oven, an RI detector, and the
Agilent Chemstation software. An IBZ Messtechnik
(Germany) Chiralyser‐MP optical rotation detector with
wavelength of 426 nm (LED) was used for detection of
isoborneol enantiomers. The VCD spectra of two isoborneol
enantiomers in CDCl3 solution were recorded on a BioTools
(Jupiter, FL) ChiralIR‐2X VCD spectrometer.
Calculated frequencies were empirically scaled using a 0.97
scaling factor.
In addition, the solid‐state IR and VCD spectra of two
enantiomers in KBr pellets were also recorded on this VCD
spectrometer. Comparison of the observed and computational
spectra details is presented in the Supporting Information
(Figures S5–S6).
Isoborneol was dissolved with n‐hexane to a concentra-
tion of 2.30 mg⋅mL−1. A mixture of n‐hexane‐alcohol was
freshly prepared and degassed in an ultrasonic bath prior to
use. The separation experiments were conducted at 283 K,
unless otherwise specified. Analyte solutions (10 μL) were
injected twice and the flow rate was 1.0 mL⋅min−1. The aver-
age and the range of the OR detector were set at off and 4.0
mdeg, respectively. Separation parameters such as retention
factor (k), selectivity (α), and resolution (Rs) were determined
according to standard formulas on the basis of USP
standards.22
3 | RESULTS AND DISCUSSION
3.1 | Screening of chiral stationary phases
Potential interactions between analyte molecules and chiral
selectors have played crucial roles in the chiral separation
process.4,5,24,25 Herein, four polysaccharide‐coated CSPs,
namely, Enantiopak OD, OX, OJ, and AD, were selected
for separation of isoborneol enantiomers by HPLC with the
mixture of n‐hexane‐ethanol (99:1, v/v) and the OR detector.
As depicted in Figure 1, isoborneol enantiomers were base-
line separated on the Enantiopak OD column, and showed
partial or no separation tendency on the others. In addition,
retention of isoborneol on the Enantiopak AD column was
much stronger than the others.
2.3 | Semipreparative separation of isoborneol
racemate
It is very difficult to predict which combination of CSP
and organic modifier would yield the effective separation
for a new chiral sample. The Enantiopak OD, OX, and OJ
columns have the same cellulose backbone, but different
derivative groups. The Enantiopak AD column contains the
same phenylcarbamate moiety as the Enantiopak OD
column, but has a different backbone. Potential forces
between isoborneol and these chiral selectors favorable to
chiral separation might be hydrogen bonds, dipole–dipole
interactions, and Van der Waals interactions. In the
Enantiopak OD CSP, intermolecular hydrogen bonds among
Semipreparative separation of isoborneol racemate with the
concentration of 50 mg⋅mL−1 was run on a semipreparative
EnantioPak OD column by HPLC with an OR detector and
the mixture of n‐hexane‐ethanol (99:1, v/v). Two enantiomer
solutions were collected separately, concentrated by rotary
evaporation, and dried in vacuo to obtain the solid samples.
Characterization results are shown in the Supporting
Information (Figures S1–S4).
2.4 | Experimental and computational IR and
VCD details
The IR and VCD spectra of two isoborneol enantiomers in
CDCl3 solution were measured at a resolution of 4 cm−1
using Synchrocell (2.75 sec per cycle) on a BioTools
ChiralIR‐2X VCD spectrometer. The CDCl3 solution of the
enantiomer was injected into a cell (150 mm in optical
length) with BaF2 windows. The ZnSe photoelastic modula-
tor of the instrument was set to 1400 cm−1. In order to
improve the signal‐to‐noise ratio, these spectra were
measured for 5 h. A baseline correction by subtracting the
solvent reference spectrum collected under the same condi-
tions was carried out for the IR spectra.
Geometry optimizations, IR and VCD spectral calcula-
tions based on the SSS configuration were performed in the
Density Functional Theory (DFT) framework with the
B3LYP hybrid functional with a 6–311 + G (2d, p) basis
set using Gaussian 09.23 The vibrational spectra were calcu-
lated for the (1S, 2S, 4S)‐isoborneol in a CHCl3 solution.
FIGURE 1 HPLC chromatograms of isoborneol enantiomers on the
four polysaccharide CSPs. Mobile phase: n‐hexane: ethanol (99:1, v/v);
the OR detector; injection volume, 10 μL; column temperature 283 K;
flow rate, 1 mL·min−1

4
GAO ET AL.
3, 5‐dimethylphenylcarbamate substituents were easily
formed and led to formation of a highly ordered secondary
structure of cellulose derivative, which further enhanced its
enantioseparation ability; in addition, long‐range electron‐
donating effects from methyl groups on 3, 5‐
dimethylphenylcarbamate moieties might enhance the basic
strength of C = O groups and helpfully form typical H‐bonds
with isoborneol molecules, further resulting in better
enantioseparation.4 The Enantiopak OX chiral selector
contained both a methyl group (electron‐donating unit) and
chlorine atom (electron‐withdrawing unit), and formation of
H‐bonds between C = O groups and hydroxyl groups of
isoborneol might be affected, which resulted in partial
separation.4 Moreover, although the Enantiopak AD chiral
selector possessed the same derivative groups as the
Enantiopak OD, a different volume of the helical groove of
the cellulose derivative and an amylose derivative led to the
different insertion of the select and in the helical cavity of
the selector.26 Thus, the Enantiopak OD column was selected
in the follow‐up work based on these screening results.
3.3 | Effects of content of alcohol
Taking into account that ethanol has been the more
commonly used modifier than n‐propanol, elution of
isoborneol in 1% ethanol was faster than the others and the
separation results of isoborneol seemed good; ethanol has
been selected to further investigate the influence of the
content (Table 2 and Supporting Figure S7). Obviously, the
retention of isoborneol showed a strengthening tendency,
with k1 ranging from 0.22 to 4.28 along with decreasing the
content of ethanol from 10% to zero. The resolution, selectiv-
ity, and theoretical plate numbers also displayed the similar
upward trend, which was due to the competitive interactions
between the solute and ethanol. The potential chiral sites of
the CSP remaining for the enantiomers increased with the
decrease of the percentage of ethanol, further resulting in
stronger retention and better enantioseparation of the
analytes.27,28 Isoborneol was better resolved in n‐hexane
(Rs, 3.20) than the others, but the elution time was much
longer and the corresponding chromatographic peaks were
broadened due to diffusion of sample molecules.
3.4 | Thermodynamic studies on enantiomeric
separation of isoborneol
3.2 | Effects of alcoholic modifiers
Composition of mobile phases has significant effects on
retention, resolution, and recognition behaviors of CSPs.
Addition of organic modifiers into mobile phase enhances
its eluting ability and improves separation of enantio-
mers.27,28 Herein, ethanol, n‐propanol, and n‐butanol were
used as the organic modifiers, respectively.
As listed in Table 1, isoborneol enantiomers were base-
line separated (Rs > 1.5) in the mixture of n‐hexane‐alcohol
(99:1, v/v). Due to different polarities of three alcohols, the
retention and resolution of isoborneol showed differences in
these mobile phases, but the separation factors remained
nearly unchanged. Among three alcoholic modifiers,
n‐propanol displayed the best separation ability with the
highest resolution. Generally, alcohol molecules and enantio-
mers were competitively adsorbed on the chiral sites of the
CSP, possibly enhancing potential H‐bonding attractions
between organic modifiers and phenylcarbamate substituents
in the CSP.
Column temperature may have a significant impact on the
adsorption–desorption equilibrium between enantiomers
and CSPs, further leading to a potentially big change of reten-
tion behaviors, resolution, and elution order of enantiomers
on CSPs. A thermodynamic study is an effective method to
investigate chiral recognition mechanism.29,30 The effect of
column temperature on separation of chiral analytes can be
expressed by Van't Hoff equations as follows29:
ΔH ΔS
ΔH
RT
lnk ¼ −
þ
þ lnΦ ¼ −
þ ΔSꢀ
(1)
RT
R
ΔΔH ΔΔS
lnα ¼ −
þ
(2)
RT
R
TABLE 2 Enantioseparation results of isoborneol under the mobile
phase with different content of ethanol*
Content of ethanol (v%)
k1
k2
α
Rs
N1
TABLE 1 Enantioseparation results of isoborneol in the normal
phase with different alcoholic modifiers*
0
4.28
5.51
1.29
3.20
10033
1
1.00
0.62
0.40
0.22
1.27
0.73
0.45
0.25
1.27
1.18
1.15
1.17
2.76
1.36
0.72
0.54
10614
11101
5965
Modifier
π
4.30
k1
k2
α
1.27
Rs
N1
Ethanol
1.00
1.27
2.76
10,614
2
n‐Propanol
n‐Butanol
4.00
3.90
1.17
1.31
1.50
1.61
1.28
1.23
3.07
2.47
10,033
9751
5
10
8856
*Chromatographic conditions: Mobile phase: n‐hexane: alcohol (99:1, v/v); the
Enantiopak OD column; the OR detector; injection volume, 10 μL; column
*Chromatographic conditions: Mobile phase: n‐hexane: ethanol (v/v); the
Enantiopak OD column; the OR detector; injection volume, 10 μL; column
temperature 283 K; flow rate 1 mL⋅min−1
.
temperature, 283 K; flow rate, 1 mL⋅min−1
.

GAO ET AL.
5
where ΔH and ΔS represent the respective changes of
enthalpy and entropy when one enantiomer transfers from a
mobile phase to the stationary phase; ΔΔH reflects the differ-
ence of interactions between both enantiomers and chiral
stationary phases; ΔΔS is defined as the difference of chaos
of enantiomers between chiral stationary phases and the
Good linear relationships between lnk and 1/T were
observed (R2 >0.99) (supporting information Table S1 and
Figure S8), indicating that the stability constant of the com-
plex formed from the CSP and the enantiomer molecule
remained unchanged in the experimental temperature range.
ΔH (ΔH1 = 4.16 kJ⋅mol−1 and ΔH2 = 6.61 kJ⋅mol−1
,
mobile phase; R is the gas constant (8.314 J⋅mol−1⋅K−1
and Φ is the column phase ratio.
)
subscripts 1 and 2 are for the first‐ and the second‐eluted
components, respectively) and ΔS* (ΔS*1 = 1.76 J⋅mol−1⋅K−1
and ΔS*2 = 2.56 J⋅mol−1⋅K−1) were obtained from the slope
and the intercept of the linear lnk vs 1/T plots. Moreover, the
ΔΔH and ΔΔS values were obtained from the differences of
ΔH and ΔS* for two enantiomers. Since both ΔΔH
(−2.45 kJ⋅mol−1) and ΔΔS (−0.80 J⋅mol−1⋅K−1) were nega-
tive, separation of isoborneol enantiomers could be ascribed
to an enthalpy‐driven process and increasing the column
temperature would lead to much more difficulty in resolution
of isoborneol enantiomers.
Separation of isoborneol has been carried out on the
Enantiopak OD column in n‐hexane‐ethanol (99:1, v/v) at
different column temperatures. As shown in Figure 2 and
Table 3, the column temperature showed obvious effects
on interactions between enantiomers and the chiral selec-
tors, that is, k1 decreased from 1.00 to 0.87 along with
increasing temperature from 283 to 308 K, and k2 followed
in a similar manner, which might be explained as a decrease
of viscosity of mobile phase with increasing temperature. In
addition, resolution and selectivity of isoborneol presented a
similar trend.
3.5 | VCD Spectra and absolute
configurations of isoborneol enantiomers
VCD is a form of chiroptical spectroscopy, which can be
used to determine absolute configurations of chiral
compounds. Both the experimental and calculated VCD
spectra of the sample are necessary. Meanwhile, if the prin-
cipal features and their signs of the experimental VCD spec-
tra show agreement with the calculated spectra, the AC of
the sample is the same as that of the structure used in the
31-35
calculation.
Figure 3 presents the experimental IR and VCD spectra
of two isoborneol enantiomers in the CDCl3 solution, and
the DFT‐calculated IR and VCD spectra of (1S, 2S, 4S)‐
isoborneol in the CHCl3 solution. Comparison of the calcu-
lated and experimental IR and VCD spectra of the (1S, 2S,
4S)‐isoborneol are depicted in Figure 4. The selected wave-
length region from 1600 cm−1 to 950 cm−1 provided rich
spectral messages for determination of AC.
The solution IR spectra of both enantiomers from prepar-
ative separation of isoborneol racemate matched well in
Figure 3A, and their VCD spectra exhibited the mirror
images of each other.
FIGURE 2 HPLC chromatograms of isoborneol on the EnantioPak
OD column under different column temperature. Mobile phase: n‐
hexane: ethanol (99:1, v/v); the OR detector; injection olume, 10 μL;
flow rate 1 mL·min‐1
As depicted in Figure 4A, the experimental VCD pattern
of the first‐eluted component was similar to the DFT calcu-
lated on the basis of SSS‐configuration. The peaks at
1521 cm−1 and 1496 cm−1 in the calculated VCD spectrum
corresponded to those at 1477 cm−1 and 1456 cm−1 in the
experimental VCD peaks, which were attributed to the
in‐plane bending vibrations of the C‐H bonds. In the experi-
mental VCD spectrum, the signals in the range from 1350 cm
TABLE 3 Effect of column temperature on separation of isoborneol*
T /K
k1
k2
α
Rs
283
1.00
1.27
1.27
2.76
288
293
298
303
308
0.97
0.94
0.92
0.89
0.87
1.22
1.16
1.12
1.06
1.01
1.25
1.23
1.21
1.19
1.17
2.61
2.41
2.08
1.92
1.75
−1
to 1100 cm−1 were caused by the bending vibrations of
C‐H bonds and stretching vibration of C‐C bonds, and
displayed a satisfactory consistency with the calculated
VCD pattern. Additionally, the bands at 1088 cm−1 and
*Chromatographic conditions: Mobile phase: n‐hexane: ethanol (99:1, v/v); the
OR detector; injection volume, 10 μL; flow rate, 1 mL⋅min−1
.

6
GAO ET AL.
FIGURE 3 (A) The experimental IR and VCD spectra of two isoborneol enantiomers in the CDCl3 solution; (B) the DFT calculated IR and VCD
spectra of (1S, 2S, 4S)‐isoborneol in the CHCl3 solution
FIGURE 4 Comparison of the observed and DFT‐calculated spectra of (1S, 2S, 4S)‐ isoborneol
1016 cm−1 in the calculated VCD spectrum were well
reproduced to the peaks at 1068 cm−1 and 999 cm−1 in the
experimental pattern related to the C–O stretching vibration.
The sign of the band at 1088 cm−1 in the calculated VCD
spectrum, or in other words, at 1068 cm−1 in the experimen-
tal presented a −/+ couplet (from lower to higher frequency),
and could be strong evidence to illustrate the similarity of the
experimental and the calculated spectra. Moreover, the exper-
imental IR spectrum of the first‐eluted component was well
consistent with the calculated pattern based on the
SSS‐configuration (Figure 4B). The experimental IR peaks
at 1390 cm−1 and 1370 cm−1 were ascribed to the bending
vibrations of the claw ring's methyl groups, which
corresponded to those calculated peaks at 1429 cm−1 and
1407 cm−1. Thus, the calculated IR and VCD spectra showed
a good agreement with the corresponding experimental
spectra, which would allow for the AC of the isoborneol
enantiomers to be reasonably assigned according to the
calculated results. It was feasible to determine that the
first‐eluted component in the HPLC separation of isoborneol
was (1S, 2S, 4S)‐isoborneol. Furthermore, taking into consid-
eration that the OR signal for the first‐eluted component was
positive in the chromatogram, it should be (1S, 2S, 4S)‐(+)‐
isoborneol. The other component was (1R, 2R, 4R)‐(−)‐
isoborneol.
4 | CONCLUSIONS
In summary, enantioseparation and semipreparation of
isoborneol were successfully conducted on the Enantiopak
OD column by normal‐phase HPLC coupled with an OR
detector. The effects of chiral stationary phases, type and
composition of organic modifiers, as well as column
temperature were investigated in detail. Two optically pure
isoborneol enantiomers were obtained by HPLC with the
OR detector, and further characterized by analytical
HPLC, multispectroscopy, and VCD theoretical calcula-
tions. The DFT‐calculated VCD spectrum based on the
SSS configuration was consistent with the observed result
of the first‐eluted component, which illustrated that the
absolute configuration of the first elution was of SSS‐form.
In brief, the proposed method described in this work is
reliable and highly effective for analysis and preparative
separation of other chiral compounds without typical
chromophores.

GAO ET AL.
7
liquid chromatography with an α‐cyclodextrin bonded stationary
phase. J Chromatogr A. 1994;666(1‐2):445‐448.
ACKNOWLEDGMENTS
Financial support from the National Natural Science
Foundation of China (Nos. 21171059, 21571070, 21603076,
and 21273175), Guangdong Provincial Science and Technol-
ogy Project (Nos. 2016B090921005, 2016B010108007 and
2014A010101145), Guangzhou Science and Technology
Project (Nos. 201508020093 and 201604020145) are grate-
fully acknowledged.
15. Mookdasanit J, Tamura H. Comparative enantioseparation of mono-
terpenes by HPLC on three kinds of chiral stationary phases with an
on‐line optical rotatory dispersion under reverse phase mode. Food
Sci Technol Res. 2002;8(4):367‐372.
16. Tian LL, Zhou Z, Zhang Q, et al. Protective effect of ( )‐isoborneol
against 6‐OHDA‐induced apoptosis in SH‐SY5Y cells. Cell Physiol
Biochem. 2007;20(6):1019‐1032.
17. Armaka M, Papanikolaou E, Sivropoulou A, Arsenakis M. Antiviral
properties of isoborneol, a potent inhibitor of herpes simplex virus
type 1. Antiviral Res. 1999;43(2):79‐92.
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SUPPORTING INFORMATION
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How to cite this article: Gao R‐Q, Fan J, Tan Q,
et al. Reliable HPLC separation, vibrational circular
dichroism spectra, and absolute configurations of
isoborneol enantiomers. Chirality. 2017;1–8. https://
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