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Comparative Enantioseparation of Seven Amino Alcohols on Teicoplanin-

Based Chiral Stationary Phases
Bao-Chun Shenab; Da-Tong Zhangac; Bei-Jia Xua; Xiu-Zhu Xua
a
Department of Chemistry, Zhejiang University, Hangzhou, P. R. China b Faculty of Pharmacy,
Kunming Medical College, Kunming, P. R. China c College of Biologic & Environmental Engineering,
Zhejiang University of Technology, Hangzhou, P. R. China

To cite this Article Shen, Bao-Chun , Zhang, Da-Tong , Xu, Bei-Jia and Xu, Xiu-Zhu(2007) 'Comparative
Enantioseparation of Seven Amino Alcohols on Teicoplanin-Based Chiral Stationary Phases', Analytical Letters, 40: 15,
2821 2839
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 Analytical Letters, 40: 28212839, 2007
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DOI: 10.1080/00032710701603801

BIOSENSORS
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Comparative Enantioseparation of Seven

Amino Alcohols on Teicoplanin-Based
Chiral Stationary Phases

Bao-Chun Shen
Department of Chemistry, Zhejiang University, Hangzhou, P. R. China
and Faculty of Pharmacy, Kunming Medical College, Kunming,
P. R. China

Da-Tong Zhang
Department of Chemistry, Zhejiang University, Hangzhou, P. R. China
and College of Biologic & Environmental Engineering, Zhejiang
University of Technology, Hangzhou, P. R. China

Bei-Jia Xu and Xiu-Zhu Xu

Department of Chemistry, Zhejiang University, Hangzhou, P. R. China

Abstract: The macrocyclic antibiotics represent a relatively new class of chiral

selectors in separation science and teicoplanin-based chiral stationary phases (CSP)
have been used successfully in a number of applications in high-performance liquid
chromatography. In the present studies, we self-prepared two bonded CSPs teicopla-
nin (TE) and teicoplanin phenyl isocyanate (TE-Phe). Seven amino alcohols, proprano-
lol, bisoprolol fumarate, atenolol, salbutamol, isoproterenol, metoprolol, and labetalol
were enantioseparated on both self-made CSPs using methanol as mobile phase and
acetic acid (HOAc) and triethylamine (TEA) as mobile phase additives. On both
CSPs, the different enantioseparation behavior of analytes with different structure was
compared. The influence of the concentration of mobile phase additives (HOAc and
TEA) on the enantioseparation was investigated. In all conditions, the retention factors
(k0 ) of seven analytes on TE-Phe CSP were larger than that on TE CSP. However, the
separation factors (a) and resolutions (Rs) on TE-Phe CSP were smaller than that on
TE CSP. The results indicated that the derivatized TE-Phe CSP is not efficient as
original teicoplanin CSP. Our observations also suggested that, for teicoplanin-based

Received 4 July 2007; accepted 15 July 2007

Address correspondence to Xiu-Zhu Xu, Department of Chemistry, Zhejiang
University, Hangzhou 310027, P. R. China. E-mail: xuxiuzhu@zju.edu.cn

2821
 2822 B.-C. Shen et al.

CSPs, p-p interactions and dipole-dipole between solutes and CSPs mainly contribute to
the retention of solutes on CSPs while hydrogen bonding and steric interactions play
important roles in the chiral recognition for teicoplanin-based CSPs.

Keywords: Teicoplanin, teicoplanin phenyl isocyanate, chiral stationary phase, amino

alcohols, enantioseparation
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INTRODUCTION

Drugs amino alcohols such as atenolol, metoprolol, propranolol, and labetalol,

etc. are chiral hydroxyl-amine-containing compounds. Some of the clinical
uses of these drugs are treatment of hypertension, anginapectoris, suprabven-
tricular, and ventricular arrhythmias and they are known to reduce the
intensity of migraine headaches. Most of the amino alcohols are marketed
as racemic mixtures. Given the fact that these drugs have many side effects
such as gastrointestinal disturbances, tiredness, dizziness, depression, par-
esthiasis, muscle aching, asthmatic wheezing and many others and that only
the levo enantiomer ((S)-enantiomer) is pharmaceutically active while the
other is either inactive or in some cases harmful, there is undoubtedly a
great need to develop quick methods for their enantiomeric resolution.
Recent experimental studies have shown that enantiomeric resolutions of
one or more amino alcohols have been successfully achieved using high-
performance liquid chromatography (HPLC). (+)-Atenolol has been resolved
using capillary elecreophoresis (Arias et al. 2001), HPLC (Lamprecht et al.
2000), and HPTLC (Bhushan and Thiongo 1998). (+)-Metoprolol has also
been resolved using HPLC (Zhang et al. 2003), while various chromatographic
methods have been used in resolving (+)-propranolol (Bhushan and Thiongo
1998). Enantioseparation of racemic amino alcohols on chiral stationary
phases (CSP) by HPLC is widely applied (Wang and Zhang 2002). Petersen
et al. (1997) has successfully enantioseparated 15 of 18 b-blocking drugs
currently on sale in Denmark using a Pirkle-type CSP, a-Burke 1 chiral
column. Zhang et al. (2005) has also successfully enantioseparated several
b-blockers on an 18-crown-6 derivative CSP and two Pirkle-type CSPs. Macro-
molecule CSPs, for examples, protein AGP (Ceccato et al. 1997), Chiralcel OD
(Aboul-Enein and Serignese 1993; Svensson et al. 1999), and b-cyclodextrin
(Crini and Morcellet 1996) have been successfully used to enantioseparate the
amino alcohols such as propranolol. Ion-pair chromatography (Xu et al. 2001),
capillary electrophoresis (Wang et al. 2000), and chiral derivatization of liquid
chromatography have also been used to separate racemic amino alcohols.
The use of macrocyclic antibiotics as chiral selectors was introduced in
1994 by Armstrong et al. (1994). The macrocyclic antibiotics are a relatively
new class of chiral selectors in separation science and have been used success-
fully to resolve a wide number of enantiomers in HPLC (Ward et al. 2001),
thin layer chromatography (Ward et al. 2001), capillary electrophoresis
 Comparative Enantioseparation of Seven Amino Alcohols 2823

(Ward et al. 2001), etc. The structure of macrocyclic antibiotics possesses

many functional groups (for example hydroxyl, amine, amide linkages, car-
boxylic acid, aromatic moieties, and hydrophobic pockets) that offer
different molecular interactions, including hydrophobic, ionic, hydrogen
bonding, dipole-dipole, p-p, and steric interactions (Ekborg-Ott et al. 1998).
The macrocyclic glycopeptide teicoplanin consists of an aglycon portion of
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fused macrocyclic rings that form a characteristic basket shape and carbo-
hydrate moieties attached to the aglycon basket. The aglycon basket consists
of three or four fused macrocyclic rings composed of linked amino acids and
substituted phenols. The carbohydrate moieties consist of carbohydrate or sac-
charide groups. Teicoplanin is unique among the glycopeptides in that it has a
hydrophobic acyl side chain attached to a 2-amino-2-deoxy-b-glucopyranosyl
group. This hydrophobic acyl side chain helps to form a hydrophobic tail;
therefore, teicoplanin is surface active and aggregate to form micelles unlike
the other glycopeptide antibiotics (Gasper et al. 1996).
The teicoplanin-based bonded phases can be used in normal-phase mode,
reversed phase mode, and polar organic mode to achieve different enantios-
electivities (Armstrong et al. 1994) or they can be derivatized to alter their
enantioselectivity. Isocyanates groups of phenyl isocyanates can react with
alcohol hydroxyl groups to form carbamate or react with amino to form urea
derivative. But phenol hydroxyl is not reactive in basic solvents such as
pyridyl, so the alcohol hydroxyl groups and amino of teicoplanin CSP were
reacted by derivation. After derivation, hydrogen-bonding capabilities are
diminished while dipole-dipole and p-p interactions are strengthened. These
changes may therefore affect the chiral discrimination on the derivatized CSP.
Teicoplanin CSP has been applied in the determination of enantiomers of
salbutamol in human plasma and urine (Joyce et al. 1998). Lamprecht et al. has
developed an HPLC column-switching method for the enantioselective deter-
mination of (R,S)-atenolol in human urine based on teicoplanin CSP
(Lamprecht et al. 2000). In the current studies, we self-prepared teicoplanin
(TE) and teicoplanin phenyl isocyanate (TE-Phe) CSPs. Isocyanate groups
were introduced for TE-Phe CSP. Seven amino alcohols were enantioseparated
on self-made TE and TE-Phe CSPs under polar organic mode. On both CSPs,
the influence of solute structure and the concentration of mobile phase
additives were investigated and the chiral recognition mechanisms were
discussed. Teicoplanin phenyl isocyanate CSP hasnt been reported and five
amino alcohols were first enantioseparated on teicoplanin-based CSPs.

EXPERIMENTAL

Materials and Chemicals

Teicoplanin was obtained from Xinchang Pharmaceutical Factory

(Xinchang, Zhejiang, P. R. China). (3-Aminopropyl) triethoxysilane (99%),
 2824 B.-C. Shen et al.

1,6-diisocyanatohexane (99%) and phenyl isocyanate (99%) were purchased

from Acros Organics (New Jersey). 3,5-dimethylphenyl isocyanate (99%)
were purchase from Aldrich. Kromasil Si 200 and Kromasil Si 100 silica
gel (5 mm) was kindly provided by Professor Dr. Kinkel as gifts. Methanol
was HPLC grade and was purchased from Ludu Chemical Reagents Plant
(Shanghai, P. R. China). Seven racemic amino alcohols, propranolol, bisopro-
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lol fumarate, atenolol, salbutamol, isoproterenol, metoprolol, and labetalol

(Fig. 1), were obtained from National Institute for the Control of Pharma-
ceutical and Biological Products (Beijing, P. R. China). All other reagents
were analytical grade reagent produced in P. R. China.

Instrumentation and Chromatographic Conditions

Elemental analysis was performed on a ThermoFinnigan Flash EA1112

CHNS analyzer). Enantioseparations were performed using Waters 2690 Sep-
arations Module equipped with a Waters 996 Photodiode Array Detector and
Waters Millennium32 System (Waters Co., Milford, MA). The mobile-phases
were methanol and containing indicated concentrations of acetic acid (HOAc)
and triethylamine (TEA) as the mobile phase additives. The flow rate of
mobile phases was set at 1 ml min21. The UV absorbance signal was
monitored at 225 nm. All chromatographic analyses were carried out at

Figure 1. The structure of seven amino alcohol drugs.

 Comparative Enantioseparation of Seven Amino Alcohols 2825

ambient temperature. The teicoplanin-based chiral columns were self-prepared

in the laboratory.

Preparation of Teicoplanin-based Chiral Columns

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Phenyl isocyanate teicoplanin was prepared by the reaction of 3.2 g teicopla-

nin with excessive phenyl isocyanate in dry pyridine for 2 h under nitrogen.
After cooling to room temperature, the derivative was filtrated and washed
sequentially with 50 ml portions of pyridine, water, methanol, and dichloro-
methane. After washing, the product was dried under vacuum.
Teicoplanin-bonded CSP was prepared according to the methods
described previously (Xu et al. 2001; Wang and Zhang 2002): After silaniz-
ation with 3-aminopropyl triethoxysilane, 3 g of dry silanized silica gel
(C, 12.26%; N, 2.56%; H, 1.74% for Kromasil Si 200) was added in 50 ml
of dry toluene with stirring using an ice-bath cooled under nitrogen, and
2.5 ml of 1,6-diisocyanatohexane was added. The mixture was heated at
708C for 2 h; after cooling to room temperature, the liquid phase was
removed by suction filtration through a filter under a nitrogen atmosphere.
The excess reactant was removed by adding another 10 ml of dry toluene.
A suspension of 1.0 g teicoplanin or phenyl isocyanate teicoplanin in
100 ml dry pyridine was added dropwise to the activated silica with stirring,
and then the mixture was heated at 708C for 12 h with continuous gentle
stirring under a nitrogen atmosphere. After cooling to room temperature,
the teicoplanin-bonded silica was isolated by filtration and washed sequen-
tially with 50 ml portions of pyridine, water, methanol, dichloromethane,
and then dried under vacuum. Elemental analysis: C, 12.26%; N, 2.56%; H,
1.74% for teicoplanin-bonded CSP and C, 12.88%; N, 2.70%; H, 1.72% for
phenyl isocyanate teicoplanin-bonded CSP, respectively.
The dried teicoplanin-based CSP powder was packed into a 300
4.6 mm
i.d. or a 250
4.6 mm i.d. stainless-steel column at 6000 psi on a Haskel air
high-pressure packing machine (Haskel Inc., Burbank) using a conventional
slurry method. A 1:1 mixture of acetone and chloroform, containing 15% of
acetic acid was used as the slurrying agent and ethanol was used as pressurizing
agent. The dead time (t0) was determined by injecting 100% ethanol. The theor-
etical plate numbers (N/m) was 6903 and 7480 for teicoplanin and phenyl
isocyanate teicoplanin chiral column, respectively for acetophenone with
n-hexane/ethanol (90:10, 1.0 ml/min) as eluent at room temperature.

Preparation of sample solutions and mobile phases

The sample solutions were prepared by dissolving solute in ethanol. The

mobile-phases were methanol with HOAc and TEA as the mobile phase
 2826 B.-C. Shen et al.

additives and were filtered through a 0.45 mm membrane filter and degassed
with an ultra-sonic bath before use.

RESULTS AND DISCUSSION

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The Influence of Structure of Amino Alcohols on Enantioseparation

The chemical structures of seven amino alcohols, propranolol, bisoprolol

fumarate, atenolol, salbutamol, isoproterenol, metoprolol, and labetalol are
similar (see Fig. 1). Except for labetalol, other amino alcohols contain one
chiral carbon. The chiral carbon connects with one hydroxyl (-OH) group
and a secondary amine group. Labetalol contains two chiral carbons but the
second chiral carbon connects directly with the secondary amine group and
no hydroxyl group is connected with this chiral carbon. Seven amino
alcohols were enantioseparated on self-made teicoplanin chiral column
under polar organic mode. The results are listed in Table 1. The results demon-
strated that all seven amino alcohols obtained enantioseparation on teicopla-
nin CSP. Figure 2 shows the enantioseparation chromatograms of seven
amino alcohols on teicoplanin chiral column. Salbutamol and isoproterenol
obtained the best enantioseparation and the separation factors (a) are 1.36
and 1.31, respectively. It is notable that these two amino alcohols contain
three hydroxyl groups. Except for the hydroxyl group connected with the
chiral carbon, isoproterenol contains two phenol hydroxyl groups and salbuta-
mol contains one phenol hydroxyl group and another alcohol hydroxyl group,

Table 1. Enantioseparation of seven amino alcohols on teicoplanin-based CSPs

Teicoplanin Teicoplanin phenyl isocyanate

Compounds k10 a Rs k10 a Rs

Propranolol 0.88 1.21 1.15 4.07 1.10 1.04

Bisoprolol 0.47 1.23 0.95 3.17 1.10 1.02
Fumarate
Atenolol 1.11 1.19 1.03 5.17 1.10 1.09
Salbutamol 0.52 1.36 1.45 1.92 1.15 1.12
Isoproterenol 0.71 1.31 1.42 2.37 1.10 0.37
Metoprolol 0.55 1.21 0.95 2.80 1.10 0.97
Labetalol 2.20a 1.29 0.90 6.85b 1.14 1.07

Chromatographic conditions: stationary phase, teicoplanin and teicoplanin phenyl

isocyanate; mobile phase, methanol/HOAc/TEA (100/0.05/0.05); flow rate, 1.0 ml
min21; detection wavelength: 225 nm.
a
The retention factor of second eluted enantiomer.
b
The retention factor of first eluted enantiomer.
 Comparative Enantioseparation of Seven Amino Alcohols 2827

suggesting that the hydroxyl groups of the solutes might play an important role
in the enantioseparation of amino alcohols on teicoplanin CSP. The retention
of isoproterenol on teicoplanin CSP is stronger than that of salbutamol,
probably due to the stronger ionic interaction and hydrogen bonding
between isoproterenol and teicoplanin CSP. It is noteworthy that labetalol
has two chiral centers, so there are four enantiomers for this compound. On
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teicoplanin column, we observed three peaks and another shoulder peak for
this compound (see Fig. 2). The separation factor between the second and
the third eluted enantiomers is comparable with the separation factor of
other amino alcohols, suggesting that the chiral carbon connects to the
hydroxyl group might have obtained better enantioseparation than another
chiral carbon.
Seven amino alcohols were also enantioseparated on self-made teicopla-
nin phenyl isocyanate (TE-Phe) CSP under polar organic mode. The results
are also listed in Table 1 and the enantioseparation chromatograms are
shown in Fig. 3. The results revealed that all seven amino alcohols also
obtained enantioseparation on TE-Phe CSP. As on teicoplanin CSP, salbuta-
mol also obtained the best enantioseparation on TE-Phe CSP and the separ-
ation factor (a) is 1.15. However, isoproterenol did not obtained good
enantioseparation on TE-Phe CSP. Actually this compound obtained the

Figure 2. The chromatograms of enantioseparation of seven amino alcohols on

teicoplanin CSP. Mobile phase: Methanol:HOAc:TEA, (100:0.05:0.10, v/v/v). other
chromatographic conditions were shown in Table 1.
 2828 B.-C. Shen et al.

worst separation on TE-Phe CSP (see Table 1 and Fig. 3). For labetalol, we
observed only 2 peaks on TE-Phe column and the peak area of the first
peak is about one third of the peak area of the second peak (see Fig. 3),
suggesting only one of the four enantiomers was enantioseparated from
other three enantiomers (Fig. 4).
The retention factors (k0 ) of seven amino alcohols on TE-Phe CSP were
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much larger than that on teicoplanin CSP. However, the separation factors
(a) and the resolution factors (Rs) of seven amino alcohols on TE-Phe CSP
are smaller than that on teicoplanin CSP. When teicoplanin was modified to
teicoplanin phenyl isocyanate (from alcohol to carbamate), the hydrogen
bonding capabilities between solutes and CSP are diminished and the

Figure 3. The chromatograms of enantioseparation of seven amino alcohols on

teicoplanin phenyl isocyanate CSP. Mobile phase: Methanol:HOAc:TEA,
(100:0.05:0.05, v/v/v). Other chromatographic conditions were shown in Table 3.
 Comparative Enantioseparation of Seven Amino Alcohols 2829

dipole-dipole and p-p interactions between solutes and CSP are strengthened.
Our observations suggested that p-p interaction and dipole-dipole between
solutes and CSPs might mainly contribute to the retention of solutes on teico-
planin-based CSPs but hydrogen bonding might contribute to the enantiose-
paration. Our results also indicated that strong retention does not mean
better separation. Salbutamol obtained the best enantioseparation on both
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CSPs (see Table 1). Isoproterenol also obtained very good enantioseparation
on teicoplanin CSP. However, this solute obtained the worst separation on
TE-Phe CSP. The hydrogen bonding, dipole-dipole and p-p interactions
between salbutamol and both teicoplanin-based CSPs are very similar with
that between isoproterenol and both CSPs. These observations suggested

Figure 4. The chromatograms of enantioseparation of seven amino alcohols on

teicoplanin phenyl isocyanate CSP. Mobile phase: propranolol, bisoprolol fumarate,
atenolol, salbutamol, isoproterenol, metoprolol, Methanol:TEA, (100:0.05, v/v). labe-
talol, Methanol:HOAc:TEA, (100:0.05:0.05, v/v/v). Other chromatographic con-
ditions were shown in Table 3.
Downloaded By: [2007-2008-2009 Kangwon National University, Samcheok Campus] At: 12:43 27

2830
Table 2. The influence of HOAc/TEA concentration in mobile phase on the enantioseparation of amino alcohols on teicoplanin-based CSPs

TE TE-Phe
Mobile phase MeOH/
Compounds HOAc/TEA (v/v/v) k10 a Rs k10 a Rs

Propranolol 100/0.05/0.05 0.88 1.21 1.15 4.07 1.1 1.04

100/0.10/0.10 0.82 1.19 1.06 2.17 1.1 0.46
100/0.20/0.20 0.77 1.18 0.92 1.29 1.1 0.32
100/0.50/0.50 0.72 1.18 0.8
Bisoprolol 100/0.05/0.05 0.47 1.23 0.95 3.17 1.1 1.02
Fumarate 100/0.10/0.10 0.43 1.22 0.81 1.61 1.11 0.44
100/0.20/0.20 0.38 1.21 0.35 0.92 1.11 0.27
100/0.50/0.50 0.35 1.22 0.23
Atenolol 100/0.05/0.05 1.11 1.19 1.03 5.17 1.1 1.09
100/0.10/0.10 1.01 1.18 0.95 2.78 1.12 0.89

B.-C. Shen et al.

100/0.20/0.20 0.91 1.17 0.92 1.65 1.11 0.48
100/0.50/0.50 0.83 1.17 0.81 0.93 1.08 0.08
Salbutamol 100/0.05/0.05 0.53 1.36 1.45 1.92 1.15 1.12
100/0.10/0.10 0.51 1.33 1.36 1.2 1.14 0.5
100/0.20/0.20 0.48 1.29 1.19 0.69 1.14 0.34
100/0.50/0.50 0.52 1.24 0.29
Downloaded By: [2007-2008-2009 Kangwon National University, Samcheok Campus] At: 12:43 27

Isoproterenol 100/0.05/0.05 0.71 1.31 1.42 2.37 1.1 0.37

Comparative Enantioseparation of Seven Amino Alcohols

100/0.10/0.10 0.69 1.29 1.28 1.51 1.09 0.17
100/0.20/0.20 0.66 1.27 1.1 0.88 1.09 0.11
100/0.50/0.50 0.62 1.26 1.13
Metoprolol 100/0.05/0.05 0.55 1.21 0.95 2.8 1.1 0.97
100/0.10/0.10 0.51 1.21 0.8 1.7 1.1 0.39
100/0.20/0.20 0.46 1.2 0.68 0.98 1.1 0.29
100/0.50/0.50 0.41 1.21 0.28
Labetalol 100/0.05/0.05 2.21a 1.29 0.9 6.85b 1.14 1.07
100/0.10/0.10 2.00a 1.3 0.93 4.19b 1.15 0.99
100/0.20/0.20 1.71a 1.31 0.94 2.51b 1.15 0.88
100/0.50/0.50 1.50a 1.36 1.1 1.34b 1.22 0.22

TE: Teicoplanin CSP; TE-Phe: teicoplanin phenyl isocyanate CSP.

Chromatographic conditions: flow rate, 1.0 ml min21; detection wavelength: 225 nm.
a
The retention factor of second eluted enantiomer.
b
The retention factor of first eluted enantiomer.

2831
 2832 B.-C. Shen et al.

that the shape of salbutamol is fit in the chiral cavity of both CSPs for effective
chiral discrimination but the shape of isoproterenol is only fit in the chiral
cavity of teicoplanin CSP, indicating that steric interactions between solutes
and CSPs play an important role in the chiral recognition for teicoplanin-
based CSPs.
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The Influence of Concentration of Additives in Mobile Phase

on Enantioseparation

To investigate the influence of HOAc and TEA additives in mobile phase on

the enantioseparation of amino alcohols on teicoplanin-based CSPs, we first
fixed the volume ratio of HOAc and TEA at 1:1 and varied the composition
of mobile phase from 100:0.05:0.05 to 100:0.50:0.50 (MeOH/HOAc/TEA,
vol/vol/vol). The results of enantioseparation of seven amino alcohols on
two teicoplanin-based CSPs are shown in Table 2. As seen in Table 2, on
both teicoplanin-based CSPs, the retention factors (k0 ) of seven amino
alcohols gradually decreased as the concentration of HOAc and TEA
increased. At the higher content of HOAc and TEA in mobile phase, the
ionic strength of mobile phase should increase. Therefore, the interaction
between the mobile phase and analytes is expected to increase and conse-
quently, the retention of analytes should decrease at the higher content of
HOAc and TEA. Also, when the volume ratio of HOAc to TEA was 1:1,
the mole ratio of HOAc to TEA was 2:1. The excess of acid was used to
protonize the solute and CSPs. As the HOAc concentration further
increased, the excess of acid was also used to increase the polarity of
mobile phase. So, as the concentration of HOAc and TEA increased, the
polarity of mobile phase increased. The increase of mobile phase polarity
then resulted in the decrease of retention of solutes on the columns. As the
concentration of HOAc and TEA in the mobile phase increased, the resolution
factors (Rs) of seven amino alcohols also gradually and significantly decreased
(see Table 2). However, the separation factors (a) of seven amino alcohols
were essentially unchanged (see Table 2). These observations indicated that
the polarity of mobile phase essentially does not affect the chiral separation.
When the composition of mobile phase was 100/0.05/0.05 (MeOH/
HOAc/TEA, vol/vol/vol), seven amino alcohols obtained the best resolution.
As discussed above, the concentration of mobile phase additives, HOAc
and TEA have great influence on the retention of amino alcohols on both
teicoplanin-based CSPs. Then we investigated the individual influence of
TEA or HOAc on the enantioseparation.
At first, with fixed amount of TEA (0.05%) in the mobile phase, the
influence of the concentration of HOAc in mobile phase on the enantiosepara-
tion of amino alcohols on two teicoplanin-based CSPs was investigated. The
data is shown in Table 3. Compare with in the absence of HOAc, there was a
tremendous decrease in the retention factors (k0 ) of the amino alcohols on both
Downloaded By: [2007-2008-2009 Kangwon National University, Samcheok Campus] At: 12:43 27

Comparative Enantioseparation of Seven Amino Alcohols

Table 3. The influence of HOAc concentration in mobile phase on the enantioseparation of amino alcohols on teicoplanin-based CSPs

TE TE-Phe
Mobile phase MeOH/
Compounds HOAc/TEA (v/v/v) k10 a Rs k10 a Rs

Propranolol 100/0/0.05 1.58 1.15 1.2 5.15 1.1 0.96

100/0.05/0.05 0.88 1.21 1.15 4.07 1.1 1.04
100/0.10/0.05 0.69 1.26 1.2 2.28 1.09 0.46
100/0.15/0.05 0.62 1.21 0.8 1.98 1.09 0.34
Bisoprolol 100/0/0.05 1 1.15 1.06 4.84 1.1 1.06
Fumarate 100/0.05/0.05 0.47 1.23 0.95 3.17 1.1 1.02
100/0.10/0.05 0.35 1.26 0.48 2.2 1.1 0.44
100/0.15/0.05 0.29 1.26 0.3 1.51 1.12 0.32
Atenolol 100/0/0.05 2.2 1.15 1.11 8.35 1.11 1.17
100/0.05/0.05 1.11 1.19 1.03 5.17 1.1 1.09
100/0.10/0.05 0.9 1.19 0.95 3.65 1.1 0.95
100/0.15/0.05 0.75 1.19 0.88 2.61 1.1 0.41
Salbutamol 100/0/0.05 2.6 1.23 2.01 7.66 1.13 1.55
100/0.05/0.05 0.53 1.36 1.45 1.92 1.15 1.12
100/0.10/0.05 0.39 1.39 1.27 1.57 1.11 0.49
100/0.15/0.05 0.34 1.37 1.07 1.11 1.1 0.33
Isoproterenol 100/0/0.05 2.22 1.22 1.74
100/0.05/0.05 0.71 1.31 1.42 2.37 1.1 0.37
100/0.10/0.05 0.53 1.35 1.2 1.52 1.09 0.27
100/0.15/0.05 0.48 1.32 1.22 1.35 1.07 0.12

2833
(continued )
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2834
Table 3. Continued

TE TE-Phe
Mobile phase MeOH/
Compounds HOAc/TEA (v/v/v) k10 a Rs k10 a Rs

Metoprolol 100/0/0.05 1.16 1.15 1.02 5.18 1.1 1.07

100/0.05/0.05 0.55 1.21 0.95 2.37 1.1 0.37
100/0.10/0.05 0.41 1.24 0.82 2.3 1.09 0.43
100/0.15/0.05 0.35 1.24 0.20 1.63 1.09 0.27
Labetalol 100/0/0.05 1.16a 1.18 0.69 3.67b 1.15 0.53
100/0.05/0.05 2.21a 1.29 0.9 6.85b 1.14 1.07
100/0.10/0.05 1.80a 1.32 0.91 4.35b 1.18 1.04
100/0.15/0.05 1.48a 1.36 0.93 3.66b 1.19 0.96

TE: Teicoplanin CSP; TE-Phe: teicoplanin phenyl isocyanate CSP.

Chromatographic conditions: flow rate, 1.0 ml min21; detection wavelength: 225 nm.
a
The retention factor of second eluted enantiomer.
b
The retention factor of first eluted enantiomer.

B.-C. Shen et al.

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Comparative Enantioseparation of Seven Amino Alcohols

Table 4. The influence of TEA concentration in mobile phase on the enantioseparation of amino alcohols on teicoplanin-based CSPs

TE TE-Phe
Mobile phase MeOH/
Compounds HOAc/TEA (v/v/v) k10 a Rs k10 a Rs

Propranolol 100/0/0.05 1.58 1.15 1.2 5.15 1.1 0.96

100/0/0.10 1.15 1.14 0.89 2.96 1.08 0.26
100/0/0.15 0.95 1.13 0.31 1.92 1.08 0.19
100/0.05/0.05 0.88 1.21 1.15 4.07 1.1 1.04
100/0.05/0.10 1.02 1.2 1.21 2.22 1.11 0.52
100/0.05/0.15 0.97 1.18 1.06 1.91 1.09 0.32
Bisoprolol 100/0/0.05 1 1.15 1.06 4.84 1.1 1.06
Fumarate 100/0/0.10 0.67 1.16 0.48 2.1 1.09 0.42
100/0/0.15 0.54 1.15 0.2 1.49 1.09 0.2
100/0.05/0.05 0.47 1.23 0.95 3.17 1.1 1.02
100/0.05/0.10 0.55 1.24 1.01 1.61 1.12 0.82
100/0.05/0.15 0.5 1.21 0.88 1.41 1.1 0.31
Atenolol 100/0/0.05 2.2 1.15 1.11 8.35 1.11 1.17
100/0/0.10 1.57 1.14 0.96 3.79 1.1 0.49
100/0/0.15 1.32 1.13 0.49 2.77 1.1 0.37
100/0.05/0.05 1.11 1.19 1.03 5.17 1.1 1.09
100/0.05/0.10 1.31 1.19 1.1 2.87 1.12 0.96
100/0.05/0.15 1.2 1.17 1.01 2.57 1.11 0.43
Salbutamol 100/0/0.05 2.6 1.23 2.01 7.66 1.13 1.55
100/0/0.10 1.84 1.28 1.7 3.84 1.12 1.18
100/0/0.15 1.58 1.2 1.42 2.78 1.11 0.93
100/0.05/0.05 0.53 1.36 1.45 1.92 1.15 1.12
100/0.05/0.10 0.68 1.32 1.52 1.24 1.16 1.03
100/0.05/0.15 0.72 1.29 1.41 1.53 1.13 0.88

2835
(continued )
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2836
Table 4. Continued

TE TE-Phe
Mobile phase MeOH/
Compounds HOAc/TEA (v/v/v) k10 a Rs k10 a Rs

Isoproterenol 100/0/0.05 2.22 1.22 1.74

100/0/0.10 1.6 1.2 1.35 2.37 1.1 0.37
100/0/0.15 1.35 1.09 1.08 1.55 1.1 0.26
100/0.05/0.05 0.71 1.31 1.42
100/0.05/0.10 0.91 1.3 1.53
100/0.05/0.15 0.94 1.25 1.3
Metoprolol 100/0/0.05 1.16 1.15 1.02 5.18 1.1 1.07
100/0/0.10 0.8 1.15 0.43 2.3 1.09 0.39
100/0/0.15 0.67 1.14 0.13 1.68 1.08 0.2
100/0.05/0.05 0.55 1.21 0.95 2.37 1.1 0.37
100/0.05/0.10 0.65 1.21 0.98 1.71 1.11 0.5
100/0.05/0.15 0.61 1.19 0.84 1.54 1.1 0.31
Labetalol 100/0/0.05 1.16a 1.18 0.69 3.67b 1.15 0.53
100/0/0.10 0.78a 1.18 0.46 1.14b 1.09 0.21
100/0/0.15 1.16a 1.18 0.69 6.85b 1.14 1.07
100/0.05/0.05 2.21a 1.29 0.9 4.20b 1.12 0.83
100/0.05/0.10 2.27a 1.31 0.96 2.32b 1.15 0.42
100/0.05/0.15 2.16a 1.3 0.91

B.-C. Shen et al.

TE: Teicoplanin CSP; TE-Phe: teicoplanin phenyl isocyanate CSP.
Chromatographic conditions: flow rate, 1.0 ml min21; detection wavelength: 225 nm.
a
The retention factor of second eluted enantiomer.
b
The retention factor of first eluted enantiomer.
 Comparative Enantioseparation of Seven Amino Alcohols 2837

teicoplanin-based CSPs when only a small amount of HOAc (0.05%) was

added into the mobile phase. However, the separation factors (a) were
increased for all seven amino alcohols. As mentioned above, the excess
acid was used to protonize the solute and CSPs. This indicates that the
chiral solutes and/or the functional groups on the teicopanin-based CSPs
can be protonated, thereby affecting charge interactions, including repulsive
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interactions between solutes and CSPs. The resolution factors (Rs) were
decreased due to the decrease of the retention factors. As the concentration
of HOAc in the mobile phase further increased from 0.05% to 0.15%, the
retention factors and also the resolution factors further decreased. However,
the separation factors (a) were essentially unchanged (see Table 3).
Then, the influence of concentration of TEA in mobile phase on the enan-
tioseparation of amino alcohols on two teicoplanin-based CSPs was investi-
gated. First, the influence of the concentration of TEA alone in mobile
phase (without HOAc additive) on the enantioseparation was investigated.
The data is shown in Table 4. In the absence of HOAc additive, as the
amount of TEA in the mobile phase increased from 0.05% to 0.15%,
the retention factors (k0 ) and the resolution factors (Rs) of the amino
alcohols on both teicoplanin-based CSPs gradually decreased. However, the
separation factors (a) were essentially unchanged, suggesting that adding
TEA in mobile phase or increasing the concentration of TEA does not
affect the essentiality of the chiral discrimination of seven amino alcohols
on both tacoplanin-based CSPs.
Next the influence of the concentration of TEA in mobile phase on
the enantioseparation was investigated with the presence of a fixed
amount of HOAc (0.05%) in the mobile phase. The results are also
shown in Table 4. Very interestingly, in the presence of 0.05% HOAc,
as the concentration of TEA in the mobile phase increased from 0.05%
to 0.10%, the retention factors (k0 ) of seven amino alcohols on teicoplanin
CSP increased instead of decreased as we observed with in the absence of
HOAc additive in the mobile phase. As the concentration of TEA in the
mobile phase further increased from 0.10% to 0.15%, the retention
factors (k) slightly decreased (Table 4). In contrast, on teicoplanin
phenyl isocyanate CSP, in the presence of HOAc, as the concentration of
TEA in the mobile phase increased from 0.05% to 0.15%, the
retention factors of seven amino alcohols gradually decreased as we
observed in the absence of HOAc additive in the mobile phase (see
Table 4).
As seen in Table 4, in the presence of 0.05% HOAc, as the concen-
tration of TEA in the mobile phase increased from 0.05% to 0.15%, the
separation factors (a) of seven amino alcohols on both teicoplanin-based
CSPs were essentially unchanged. The resolution factors (Rs) of seven
amino alcohols on teicoplanin CSP were also essentially unchanged.
However, the resolution factors (Rs) of seven amino alcohols on teicoplanin
phenyl isocyanate CSP were gradually decreased (see Table 4).
 2838 B.-C. Shen et al.

CONCLUSION

Propranolol, bisoprolol fumarate, atenolol, salbutamol, isoprenaline, metopro-

lol and labetalol were enantioseparated under polar organic mode on teicopla-
nin and teicoplanin phenyl isocyanate CSPs. Enantioseparation mechanisms
were investigated and different behavior of seven amino alcohols on both
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CSPs was compared. Propranolol, atenolol, and Labetalol have stronger

retention than salbutamol on teicoplanin CSP, but they didnt obtain better
enantioseparation. The retention factors (k0 ) of seven amino alcohols on teico-
planin phenyl isocyanate CSP were larger than that on teicoplanin CSP.
However, the enantioseparation of seven amino alcohols on teicoplanin CSP
was better than that on teicoplanin phenyl isocyanate CSP. Our observations
indicated that stronger rentention doesnt mean better separation. Our obser-
vations also suggested that p-p interaction and dipole-dipole between
solutes and CSPs might mainly contribute to the retention of solutes on
CSPs while hydrogen bonding and steric interactions play important roles in
the chiral recognition for teicoplanin-based CSPs.

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