2
TANG ET AL.
simultaneously determine the ee values for many chiral
compounds in a complex sample.
In this study, ee is defined as the difference between
the enantiomeric fractions (EFs) of two enantiomers:
selection for UV and CD detection, (b) trace deviations
from Beer's law in their UV and CD signals with increas-
ing compound mass, (c) estimate the influence of these
deviations on the determination of ee, (d) establish a non-
linear relationship between the UV and CD signals, and
13
ee ¼ EF2 − EF1:
(1)
(
e) apply the new functions to calculate ee values for sam-
ples and evaluate the accuracy. This is the first time that
the deviations from Beer's law are observed in both UV
and CD detection and considered for CD‐related analysis.
In solutions, the EF of each enantiomer is equal to the
concentration of one enantiomer divided by the total con-
centration of the chiral substance. Accordingly, ee can be
expressed as
2
| MATERIALS AND METHODS
.1 | Chemicals and materials
ee ¼ ðc2 − c1Þ=ðc1 þ c2Þ;
(2)
2
where c and c are the concentrations of two enantio-
1
2
mers. For optical detectors, (c + c ) correlates to the
UV absorption signal (UV), while the absolute value for|
1
2
Racemic napropamide (1), lactofen (2), diclofop‐methyl
3), and myclobutanil (4) (all purities >95%) were
(
c ‐c |can be seen as the concentration excess of the dom-
2
1
obtained from Kuaida Agrochemical Co., Ltd. (Jiangsu,
China), Nutrichem Co., Ltd. (Zhejiang, China), Yifan
Biotechnology Group Co., Ltd. (Zhejiang, China), and
Changshu Hengrong Commerce Co., Ltd. (Jiangsu,
China), respectively. HPLC‐grade solvents, including
hexane (Hex), ethanol (EtOH), and isopropanol (IPA),
were purchased from Sigma‐Aldrich, Shanghai, China.
The semipreparative chiral columns (10 × 250 mm,
inant enantiomer and correlates to the CD signal. There-
fore, ee can be measured by UV and CD detection. If
Beer's law (the Bouguer‐Lambert‐Beer Law) is valid and
both the UV and CD signals are in linear correlation with
the concentration, ee can be determined by
ee ¼ k·CD=UV;
(3)
5
μm), including Chiralcel AD‐H, AS‐H, OD‐H, and
where CD and UV refer to the CD and UV signals, and
k is a constant.
OJ‐H, were purchased from Daicel Chiral Technologies,
China. A normal‐phase column packed with bare silica
gel (4.6 × 250 mm, 5 μm) was purchased from Welch
Materials, Inc. (Shanghai, China).
Beer's law linearly correlates the absorbance to the
thickness of the path length and the concentration of
the material sample. It has been unquestionably applied
to analytical methods related to optical detectors.
However, many studies have found that Beer's law only
works for analytes with low concentrations and that a
nonlinear relationship may exist between the absorbance
and the concentration when the analytes are at high‐
2.2 | Instruments
Enantiomeric separations of the racemates were run on a
Waters semipreparative HPLC system (Milford, MA,
USA), including a quaternary gradient module, an
autosampler with a 100‐μL loop, a column heater, a photo-
diode array (PDA) detector, and a fraction collector (mod-
ules 2535, 2707, 1500, 2998, and WFC III, respectively).
The HPLC system was controlled using the Empower soft-
ware. A Jasco UV‐Visible spectrophotometer (V‐750) with
a 1‐cm quartz cuvette was used to record the UV spectra of
compounds 1 to 4. Unitless UV signals (absorbance) were
obtained and converted into epsilon (M cm ). A Jasco
CD spectrometer (J‐1500) with a 1‐mm quartz cuvette
was used to record the CD spectra of the enantiomers.
The CD signals in the unit of millidegrees (mdeg) were
obtained and converted into delta‐epsilon (M cm ).
The analyses of compounds 1 to 4 for ee measurements
were performed on a Jasco analytical HPLC system
consisting of an autosampler with a 100‐μL loop, a quater-
nary pump, a column oven, a PDA detector, and a CD
detector (modules AS‐4050, PU‐4180, CO‐4061, MD‐
14,15
concentration levels.
These deviations may be
ascribed to the occurrence of fluorescence, scattering,
physical interactions or chemical equilibrium, the refrac-
1
4-18
tive index of the solvent, and many other effects.
The
errors introduced by using Beer's law without correction
can easily exceed one order of magnitude, so the limits
should be clearly understood before applying Beer's law
19
for any absorbance‐related analysis. Because of the
low sensitivity of CD detectors for CD detection, a high
concentration is often necessary to acquire sufficient CD
signals for ee analysis. Therefore, deviations from Beer's
law should be carefully monitored and fully considered
to determine the ee more accurately.
−
1
−1
−
1
−1
In this study, the enantiomers of four chiral pesticides,
including napropamide (1), lactofen (2), diclofop‐methyl
(
3), and myclobutanil (4) were separated from commer-
cial racemates and used to (a) obtain the UV and CD
spectra for chiral characterization and wavelength