SEPARATIONS—b-ADRENERGIC AGONISTS AND ANTAGONISTS
25
TABLE 1. The retention, separation, and resolution factors
EXPERIMENTAL
Chemicals and Reagents
of chiral separation of b-blockers on AmyCoat column
Sl. no.
b-Blockers
k1
k2
a
Rs
n-Hexane, ethanol, methanol, and diethylamine were of
HPLC grade and obtained from E. Merck, India. All other
chemicals and reagents were of analytical grade and were
purchased from E. Merck, India. Sixteen b-adrenergic
blockers namely acebutalol, alprenolol, atenolol, bisopro-
lol, bopindolol, bufurolol, carazolol, celiprolol, cimeterol,
clenbuterol, indenolol, metaprolol, nebivolol, oxprenolol,
practolol, propranolol, tertalol, and timolol and two adre-
nergic agonists, viz., cimeterol, clenbuterol, were pur-
chased from Sigma (St. Louis, MO) or were donated gifts
from other departments.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Acebutalol
Alprenolol
Atenolol
0.74
0.38
1.12
0.99
0.96
5.53
2.53
3.50
2.61
2.31
0.47
1.52
10.91
0.76
1.88
9.50
0.67
6.80
0.80
0.54
1.56
1.21
2.10
6.30
3.74
8.70
4.24
4.20
0.79
1.91
16.72
1.14
2.18
19.60
0.95
9.40
1.08
1.42
1.39
1.22
2.18
1.14
1.48
2.33
1.62
1.85
1.66
1.26
1.53
1.53
1.59
1.10
1.42
1.33
1.87
1.08
2.95
1.32
3.50
1.00
2.19
4.50
2.85
3.20
1.87
1.70
2.00
1.84
1.00
1.10
1.54
2.06
Bisoprolol
Bopindolol
Bufurolol
Carazolol
Celiprolol
Cimeterol
Clenbuterol
Indenolol
Metaprolol
Nebivolol
Oxprenolol
Practolol
HPLC Analysis
All HPLC experiments were carried out on an HPLC
system of ECOM (Czech Republic) consisting of solvent 15
16
17
18
Propranolol
Tertalol
Timolol
delivery pump (model Alpha 10), manual injector, absorb-
ance detector (Sapphire 600 UV-vis.), Chromatography I/
F module data integrator (Indtech. Instrument, India), and
Winchrome software. Chiral column used was AmyCoat
(150 3 46 mm, 3-lm silica particle size; a gift from Kroma-
sil, Eka Chemicals Separation Products, Bohus, Sweden).
The stock solutions (0.1 mg/ml) of the racemic b-adrener-
gic antagonists and agonists used in this study were pre-
pared in methanol. Injection of 5.0 ll of each solution was
injected on to the HPLC system described above. The mo-
bile phases used in this study were different combinations
of n-heptane-ethanol-diethyl amine at various flow rates
(Table 1). The mobile phases were filtered and degassed
before use daily. The separations were carried out at room
temperature with detection at 225 nm. The chromato-
graphic parameters such as capacity (k), separation (a),
and resolution (Rs) factors were calculated.
Chromatographic Conditions:
Mobile phase:
Column:
Flow rate:
Detection:
n-Heptane-ethanol-diethylamine (85:15:0.1, v/v/v).
AmyCoat (150 3 46 mm, 3 lm size silica particle).
1.0 ml/min.
UV, 225 nm.
Sample volume: 5 ll of 0.1 mg/ml concentrations.
Temperature:
Exceptions:
Flow rates:
(27 6 1)8C.
Bufurolol: 0.5, Acebutalol, Clenbuterol: 2.0, and Nebivolol: 3.0 ml/min
flow rates, respectively.
Mobile phase:
Bufurolol, Clenbuterol, Timolol: (95:05:0.1, v/v/v) and Tertalol: (90:10:0.1,
v/v/v) of n-Heptane-ethanol-diethylamine, respectively.
found to be a suitable flow rate for the resolution of acebu-
talol and clenbuterol racemates. The maximum resolution
achieved was of celiporolol (Rs 5 4.50), whereas the mini-
mum separation was of bufurolol (Rs 5 1.00). The effect of
diethylamine was investigated on chiral resolution and it
was observed that peaks had become sharp using n-hep-
tane-ethanol-diethylamine (90:10:0.2, v/v) and (90:10:0.3,
v/v) mobile phases. However, the limits of detection
decreased greatly by increasing the concentrations of
diethylamine. Similarly, the polarity of the mobile phase
was also varied using 5.0, 10.0, and 15.0 ml of ethanol. As
a result of exhaustive experimentation, the chromato-
graphic conditions were varied and optimized and the best
ones are reported herein.
RESULTS AND DISCUSSION
Chiral Separations
The chromatographic parameters k, a, and Rs for the
resolved enantiomers of racemic analytes used in this
study are given in Table 1. The values k, a, and Rs ranged
from 0.38–19.70, 1.08–2.33, and 1.0–4.50, respectively. The
values of the Rs for all the racemates are greater than one,
which indicate the base line separation. The peaks were
sharp for most of the b-blockers except for clenbuterol
and timolol, which show slightly broad peaks. The chro-
matograms of celiprolol (Rs 5 4.50) and bufurolol (Rs 5
1.00) are shown in Figures 1 and 2, respectively.
Most of the b-adrenergic antagonists and agonists were
resolved using n-heptane-ethanol-diethylamine (85:15:0.1,
v/v/v) as mobile phase. However, the best separation of
tertalol was achieved using of n-heptane-ethanol-diethyl-
Chiral Recognition Mechanism
These results can be explained on the basis of the chiral
amine (90:10:0.1, v/v) as mobile phase. On the other recognition mechanisms as described by Aboul-Enein and
hand, bufurolol, clenbuterol, and timolol resolved better Ali.2 Basically, amylose has well-defined grooves providing
using (95:05:0.1, v/v/v) mobile phase consisting of n-hep- the chiral surface to the enantiomers. The retention of
tane-ethanol-diethylamine. Similarly, common flow rate enantiomers is controlled by p-p interactions, hydrogen
was 1.0 ml/min for most racemates but bufurolol and nebi- bondings, dipole induced dipole interactions, steric effects,
volol were resolved better at 0.5 and 3.0 ml/min. flow and other interactive forces. It may be concluded that b-ad-
rates, respectively. On the other hand, 2.0 ml/min was renergic blockers having strong bindings to the CSP got
Chirality DOI 10.1002/chir