Ultrasound-Controlled Chiral Separation of Four Amino Acids and 2,2,2-Trifluoro-1-(9-anthryl)ethanol

Article abstract of DOI:10.1002/bkcs.11659

Chiral separation of 4-hydroxyphenylglycine, phenylglycine, tryptophan, methionine, and 2,2,2-trifluoro-1-(9-anthryl)ethanol (TFAE) was performed under ultrasound reduction at room temperature and high temperature (50 °C). At high temperature (50 °C), both α and Rs were improved slightly under ultrasound reduction as compared to those under non-ultrasonic and ultrasonic irradiation (50 watt/L) conditions. Even at low temperatures, the largest α was observed under ultrasound reduction conditions, except in the case of methionine. However, at low temperature, Rs was reduced under ultrasound (50 watt/L) irradiation, but was improved under ultrasound reduction rather than under the continuous ultrasonic irradiation. Similar to the fact that gradient elution (based on solvent polarity) can improve α, ultrasound reduction can improve α and Rs. Ultrasound reduction is demonstrated to aid the rapid separation of chiral compounds with improved resolution, especially, at high temperatures. Although chromatographic separation using ultrasound has been rarely dealt with until now, ultrasound can be used as an external field in chromatography.

Full text of DOI:10.1002/bkcs.11659

Article
DOI: 10.1002/bkcs.11659
BULLETIN OF THE
J. H. Lee and J. J. Ryoo
KOREAN CHEMICAL SOCIETY
Ultrasound-Controlled Chiral Separation of Four Amino Acids and
2,2,2-Trifluoro-1-(9-anthryl)ethanol
*
Jae Hwan Lee and Jae Jeong Ryoo
Department of Chemistry Education, Kyungpook National University, Daegu, South Korea, 702-701.
*E-mail: jjryoo@knu.ac.kr
Received November 21, 2018, Accepted December 5, 2018, Published online January 28, 2019
Chiral separation of 4-hydroxyphenylglycine, phenylglycine, tryptophan, methionine, and 2,2,2-trifluoro-
1-(9-anthryl)ethanol (TFAE) was performed under ultrasound reduction at room temperature and high
temperature (50 ꢀC). At high temperature (50 ꢀC), both α and Rs were improved slightly under ultrasound
reduction as compared to those under non-ultrasonic and ultrasonic irradiation (50 watt/L) conditions.
Even at low temperatures, the largest α was observed under ultrasound reduction conditions, except in the
case of methionine. However, at low temperature, Rs was reduced under ultrasound (50 watt/L) irradia-
tion, but was improved under ultrasound reduction rather than under the continuous ultrasonic irradiation.
Similar to the fact that gradient elution (based on solvent polarity) can improve α , ultrasound reduction
can improve α and Rs. Ultrasound reduction is demonstrated to aid the rapid separation of chiral com-
pounds with improved resolution, especially, at high temperatures. Although chromatographic separation
using ultrasound has been rarely dealt with until now, ultrasound can be used as an external field in
chromatography.
Keywords: Ultrasound, Chiral separation, HPLC
Introduction
enantioseparation process combining the CSP method with
the CMPA method was closely examined.15
In order to efficiently separate various chiral samples, it is
highly important to select appropriate mobile phase and sta-
tionary phase suitable for each analyte. Many efforts have
been made to improve separation factor and reduce analysis
time on liquid chromatography.1–5 Enantioseparation with
comparatively high resolution of sotalol as a medicine for
treatment of complex ventricular arrhythmias has success-
fully achieved either by increasing or decreasing tempera-
ture.6 Loun also showed that the temperature changes
improved chiral separation on a protein-based chiral sta-
tionary phase.7
Ultrasound is widely used in the fields of medicine, phar-
macology, cosmetics, food and various industries. Many
studies have been conducted by using ultrasound with chro-
matography to date so as to improve the extraction effi-
ciency in the sample preprocessing stage.8–11 Okada first
attempted to investigate the influence of ultrasound on the
retention and selectivity of some ions by performing direct
ultrasound irradiation on the column in ion-exchange chro-
matography.12,13 Our research team also introduced ultra-
sound for the first time in chiral chromatography.14,15 At
high temperature, ultrasound remarkably enhanced the effi-
ciency of chiral separation and reduced the analysis time.
We termed this ultrasound assisted chromatographic tech-
nique as sonochromatography.14 In a follow-up study, the
influence of temperature and ultrasonication during the
The frequency of the ultrasonic waves used in our previ-
ous two studies conducted by our team is 42 kHz. Ultra-
sonic equipment using this frequency is widely used for
washing glassware and instruments attached to laboratory
glassware and laboratory equipment, and the cavitation
strength is low.16 In this study, chiral separation is per-
ꢀ
formed at 25 and 50 C where the strength of ultrasonic
cavitation is six to eight times higher than that in our previ-
ous studies. The shortest retention time starting from the
first peak for each sample practically corresponded to a
watt density of 50 watt/L. Thus, until the first peak
appeared, ultrasonic waves were irradiated at this watt den-
sity. As soon as the first peak started elution, ultrasonic
power was switched off; the second peak was eluted later
than that of continuous ultrasonic radiation, so that the sep-
aration factor and resolution were expected to be improved
by turning the ultrasound off.
Experimental
Instrument and Reagents. HPLC analysis was carried out
on a Jasco Pu-2080 system (Jasco, Tokyo, Japan), consist-
ing of a Plus Intelligent HPLC pump, Rheodyne Model
7725i injector (Rheodyne, Rohnert Park, CA, USA) with
sample loop 20 μL, and UV-2075 Plus Intelligent UV/Vis
detector. All chromatogram were obtained and calculated
for retention time and peak width by BORWIN
Bull. Korean Chem. Soc. 2019, Vol. 40, 146–149
© 2019 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Wiley Online Library
146

Article
BULLETIN OF THE
ISSN (Print) 0253-2964 | (Online) 1229-5949
KOREAN CHEMICAL SOCIETY
NH2
OH
HPLC and ultrasound experiment. In order to simulta-
neously carry out HPLC experiments with ultrasound irra-
diation at 25 and 50 ꢀC, a column support frame was
installed so that each column was maintained at a constant
height in the ultrasonicator tank. The column was coiled
with a PTFE tube around it. The temperature was kept con-
stant at the desired level by preventing ultrasound-induced
temperature changes with the help of a cooling circulator
that allowed an ethanol/H2O mixture to flow into the PTFE
tube. Prior to the column connection, the apparatus was
washed with methanol, and the mobile phase was allowed
to flow into the HPLC system to stabilize the system.18,19
After stabilization, each column was subjected to ultra-
sound irradiation at 25 and 50ꢀC. The analysis conditions
for each sample are as follows.
NH2
OH
O
O
HO
Phenylglycine
4-Hydroxy-phenylglycine
O
HN
H2 N
S
OH
H3 C
OH
NH2
Methionine
O
Tryptophan
HO
CF3
4-Hydroxyphenylglycine: column; Chirosil®RCA(+),
flow rate; 1.0 mL/min, detection; 210 nm, mobile phase;
methanol/water (50:50) + 1 mM ammonium acetate +
10 mM perchloric acid. Phenylglycine: column; Chiro-
sil®RCA(+), flow rate; 1.0 mL/min, detection; 210 nm,
mobile phase; methanol/water (50:50) + 1 mM ammonium
acetate + 10 mM perchloric acid. Tryptophan: column;
Chirosil®RCA(+), flow rate; 1.5 mL/min, detection;
210 nm, mobile phase; methanol/water (70:30) + 10 mM
acetic acid. Methionine: column; Chirosil®RCA(+), flow
rate; 1.0 mL/min, detection; 210 nm; mobile phase; metha-
nol/water (45:55) + 10 mM acetic acid. TFAE: column; C3
symmetric CSP,17 flow rate; 1.2 mL/min, detection;
254 nm, mobile phase; 10% IPA in hexane.
2,2,2-Trifluoro-1-(9-anthryl)ethanol (TFAE)
Figure 1. Chiral samples used in this study.
chromatography software version 1.5 (JASCO, Tokyo,
Japan). The chiral columns used are as follows, with an
inner diameter of 4.6 mm and length of 25 cm: cellulose
column, Lux 5μm cellulose-1, Phenomenex Inc., Torrance,
CA, USA; crown ether column; ChiroSil®RCA(+), Regis
Technologies, Inc., Grove, IL, USA. C3 symmetric CSP;
home-made CSP.17 All the samples used in this experiment
were purchased from Sigma Aldrich Inc. (Milwaukee, WI,
USA). The structures of the samples used in this work are
Under the ultrasound reduction, the sample was analyzed
at a watt density of 50 watt/L during column separation,
and the ultrasonicator was turned off right after the elution
of the first peak started so that the watt density was
shown in Figure 1.
4-Hydroxyphenylglycine at 50°C
HPLC solvents, acetonitrile, n-hexane, isopropyl alcohol
(IPA), ethanol, and methanol (HPLC grade) were obtained
from J. T. Baker (Center Valley, PA, USA) and mixed at a
constant ratio. Diethylamine used as the mobile phase mod-
ifier in the cellulose column was purchased from Sigma
Aldrich Inc. For the Chirosil column, perchloric acid, sulfu-
ric acid, and acetic acid used as the acidic additives
(JUNSEI grade reagents, Chuo-ku, Tokyo, Japan). Deio-
nized (DI) water used as the solvent, with a conductivity of
1 μs/cm or less, and was obtained using a MILLI-Q water
system (Bedford, MA, USA). An S8525-12 ultrasonicator
(BRANSON) was used in this study. The input power was
200 V (Æ 15%) 50/60 Hz, while the power output was
500 W, 25 Hz. In order to ensure the same analytical condi-
tions during the experiment, ultrasound was applied by
installing an HPLC column support frame in an ultrasonic
tank (CH1012-25-12) filled with the same amount of water
(10 L). An MRC-103RPD refrigerated bath circulator was
connected to the inside of the ultrasonicator to maintain the
bath temperature and thus minimize any ultrasound-induced
temperature change.
1200000
1000000
800000
600000
400000
200000
0
1200000
1000000
800000
600000
400000
200000
0
2.7
-200000
2.9
3.1
3.3
3.5
3.7
-200000
nonsonic
reduction
50watt/L
0 watt/L (nonsonic)
50 watt/L
1500000
1000000
500000
0
1500000
1000000
500000
0
0
1
2
3
4
5
0
1
2
3
4
5
-500000
-500000
Time(min)
Time(min)
Reduction (gradient)
1500000
1000000
500000
0
0
1
2
3
4
5
-500000
Time(min)
Figure 2. Chromatograms of 4-hydroxyphenylgycine at 50 ꢀC
under respective ultrasonic conditions. Mobile phase; methanol:
water (50:50)+1mM ammonium acetate+10mM perchloric acid,
flow rate; 1.0 mL/min, detection; 210 nm, column; Chirosil®
RCA(+).
Bull. Korean Chem. Soc. 2019, Vol. 40, 146–149
© 2019 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de
147

Ultrasound-Controlled Chiral Separation of Four Amino Acids and
BULLETIN OF THE
Article
2,2,2-Trifluoro-1-(9-anthryl)ethanol
KOREAN CHEMICAL SOCIETY
0 watt/L. The results were compared with those obtained
under ultrasonic irradiation at a watt density of 50 watt/L
throughout the elution time and under non-ultrasonic condi-
tions (watt density: 0).
we analyzed the other four samples and the results are sum-
marized in Table 1 below.
As shown in Table 1, the separation factor (α) and res-
olution (Rs) were slightly improved at the high tempera-
tures (50 ꢀC) under ultrasound reduction. Even at low
temperatures, the largest α was observed under ultra-
sound reduction conditions, except in the case of methio-
nine. However, at low temperature, Rs was reduced
under ultrasound (50 watt/L) irradiation, but was
improved under ultrasound reduction rather than under
the continuous ultrasonic irradiation. Similar to the fact
that gradient elution (based on solvent polarity) can
improve α, ultrasound reduction can improve α and Rs. It
has been confirmed that ultrasound can be applied to
obtain the optimal conditions for achieving the best sepa-
ration factor and resolution along with reduction of anal-
ysis time. Ultrasound irradiation at low temperature
resulted in a shorter retention time than that under non-
ultrasonic conditions, as was evident from the compari-
son of the retention time (tR1) of the first eluting peak.
At high temperature, the phenomenon is similar to that at
low temperature, but it is small. Hence, chiral separation
of 4-hydroxyphenylglycine, phenylglycine, tryptophan,
Results and Discussion
First, 4-hydroxyphenylglycine was applied to non-sonic
(watt density 0 watt/L), watt density 50 watt/L, and ultra-
sound reduction (watt density 50 to 0 watt/L) at 50 ꢀC
using a Chirosil® RCA(+) column as a stationary phase.
The chromatograms obtained from under each condition
are shown in Figure 2.
As shown in Figure 2, the elution time of the first peak
was the same at 2.91 min under ultrasound reduction (green
color, small dotted line) and ultrasonic condition (blue
color, solid line) but relatively slowed under the non-
ultrasonic condition, 2.93 min (red color, large dotted line).
The elution order of the second peak was in order of sonic,
ultrasound reduction and non-sonic. In fact, 2.91 and
2.93 min are the values within the error range. However,
relatively comparing three different types (non-ultrasonic,
ultrasonic and ultrasound reduction) of experiments, it
always shows this pattern and reproducibility is as high as
nearly 100%. Therefore, we can see that the difference
between the first peak and the second peak was the longest
under ultrasound reduction compared to ultrasonic and non-
ultrasonic conditions and the largest value of separation
factor was shown under ultrasound reduction. In this way,
methionine,
and
2,2,2-trifluoro-1-(9-anthryl)ethanol
(TFAE) was performed under ultrasound reduction at
ꢀ
room temperature and high temperature (50 C). At high
temperature (50 ꢀC), both α and Rs were improved
slightly under ultrasound reduction as compared to those
under non-ultrasonic and ultrasonic irradiation
(50 watt/L) conditions. Even at low temperature,
Table 1. Influence of ultrasound on enantioseparation of five chiral samples.
25 ꢀC
50 ꢀC
Sample
Sonication (watt/L)
tR1 (min)
α
Rs
tR1 (min)
α
Rs
4-Hydroxyphenylglycine
0
50
3.57
3.57
3.57
3.64
3.6
3.59
7.34
7.3
3.14
3.14
3.16
2.84
2.89
2.92
1.57
1.56
1.58
1.81
1.77
1.76
1.37
1.37
1.38
2.92
2.74
2.86
2.81
2.69
2.71
1.89
1.79
1.81
1.37
1.32
1.32
1.93
1.77
1.79
2.93
2.91
2.91
2.95
2.93
2.94
5.74
5.75
5.75
3.56
3.55
3.55
8.54
8.51
8.58
2.56
2.72
2.88
2.4
1.25
1.25
1.29
1.25
1.26
1.27
0.96
0.98
0.98
0.83
0.85
0.85
1.18
1.24
1.25
Reduction
0
50
Reduction
0
50
Reduction
0
50
Reduction
0
50
Phenylglycinea
2.56
2.56
1.22
1.22
1.22
1.44
1.47
1.47
1.28
1.28
1.28
Tryptophanb
7.3
Methioninec
4.08
4.16
4.15
12.69
11.84
11.52
2,2,2-Trifluoro-1-(9-anthryl)ethanol (TFAE)e
Reduction
a
Flow rate; 1.0 mL/min, detection; 210 nm, mobile phase; methanol: water(50:50) + 1 mM ammonium acetate+10 mM perchloric acid, column;
Chirosil® RCA(+).
b
c
d
Flow rate; 1.5 mL/min, detection; 210 nm, mobile phase; methanol: water(70:30) + 10 mM acetic acid, column; Chirosil® RCA(+).
Flow rate; 1.0 mL/min, detection; 210 nm, mobile phase; methanol: water(45:55) + 10 mM acetic acid. Column; Chirosil® RCA(+).
Flow rate; 1.0 mL/min, detection; 210 nm, mobile phase; methanol: water(50:50) + 1 mM ammoniumacetate+10 mM perchloric acid, column;
Chirosil® RCA(+).
e
Flow rate; 1.2 mL/min, detection; 254 nm, mobile phase; 10% IPA in hexane, column; C3 symmetric CSP.17
Bull. Korean Chem. Soc. 2019, Vol. 40, 146–149
© 2019 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de
148

Ultrasound-Controlled Chiral Separation of Four Amino Acids and
BULLETIN OF THE
Article
2,2,2-Trifluoro-1-(9-anthryl)ethanol
KOREAN CHEMICAL SOCIETY
ultrasound reduction was more effective for separation
than the non-ultrasonic conditions. Whether low or high
temperature or not, the values (α and Rs) of all samples
except methionine are within error ranges but α has
shown good results under both continuous and reduction
ultrasonic conditions and especially at high temperatures.
Under ultrasound reduction, α and Rs has shown the
largest values at high temperatures. As explained earlier
about Figure 2, the difference of the values of α and Rs
are small but consistent. The values of α and Rs calcu-
lated are within error range and even peak shapes are bad
magnifying the peaks. However, it is certainly true that
there is an effect of ultrasound. We have not done studies
under any ultrasound reduction on all samples, but we
can see that ultrasonic waves can also be examined in an
ultrasound-controlled manner as gradient elution is used
to improve separation factors. Therefore, the analysis
time and separation efficiency can be improved by exam-
ination of the ultrasound reduction.
Acknowledgments. This work has been supported by a
grant from the Korean Research Foundation (KRF)
(2016R1D1A1B03935000).
References
1. B. Yan, J. Zhao, J. S. Brown, J. Blackwell, P. W. Carr, Anal.
Chem. 2000, 72, 1253.
2. W. Lee, D. Cho, B. O. Chun, T. Chang, M. Ree,
J. Chromatogr. A 2001, 910, 51.
3. M. H. Chen, C. Horvath, J. Chromatogr. A 1991, 788, 51.
4. H. Kanazawa, Y. Kashiwase, K. Yamamoto, Y. Matsushima,
A. Kikuchi, Y. Sakurai, T. Okano, Anal. Chem. 1997, 69, 823.
5. V. Gerd, S. Pat, J. Sep. Sci. 2006, 29, 1822.
6. K. Flude, A. W. Frahm, J. Chromatogr. A 1999, 858, 33.
7. B. Loun, D. S. Hage, Anal. Chem. 1994, 66, 3814.
8. J. Shen, X. Shao, Anal. Chim. Acta. 2006, 561, 83.
9. J. Pan, X. X. Xia, J. Liang, Ultrason. Sonochem. 2008,
15, 25.
Conclusion
10. P. Bhandari, N. Kumar, B. Singh, V. K. Kaul, J. Chromatogr.
A 2008, 1194, 257.
Ultrasound reduction is demonstrated to aid the rapid sepa-
ration of chiral compounds with improved resolution, espe-
cially, at high temperatures. Although chromatographic
separation using ultrasound has been rarely dealt with until
now, ultrasound can be used as an external field in chroma-
tography. Effect of applying ultrasonication on chiral sepa-
ration is not clear at present as changes in selectivity and
resolution for the analaytes studied upon application sonica-
tion is not large enough to clearly demonstrate the effect. It
is thought that a wider variety of chiral analytes having
very high separation factor (α) than amino acids should be
investigated in order to confirm the beneficial effect of
ultrasound on chiral separation.
11. J. H. Mehdi, P. Hadi, E. N. Heshmatollah, J. Chromatogr. A
2009, 1216, 6088.
12. T. Okada, J. Chromatogr. A 1998, 793, 365.
13. S. Oszwaldowski, T. Okada, J. Chromatogr. A 1999, 850, 9.
14. J. J. Ryoo, Y. A. Song, Y. H. Jeong, M. H. Hyun, J. H. Park,
W. J. Lee, Bull. Korean Chem. Soc. 2006, 27, 637.
15. J. H. Lee, J. J. Ryoo, Bull. Korean Chem. Soc. 2012,
33, 4141.
16. J. H. Sun, S. P. Sun, J. Y. Sun, R. X. Sun, L. P. Qiao,
H. Q. Guo, M. H. Fan, Ultrason. Sonochem. 2007, 14, 761.
17. J. J. Yu, D. W. Armstrong, J. J. Ryoo, Chirality 2018, 30, 74.
18. https://www.emerson.com/documents/automation/brochure-
ultrasonic-cleaning-primer-en-5169644.pdf.
19. http://www.registech.com/chiral-applications.
Bull. Korean Chem. Soc. 2019, Vol. 40, 146–149
© 2019 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de
149