Enantiomeric separation of β-blockers and tryptophan using heparin as stationary and pseudostationary phases in capillary electrophoresis

Article abstract of DOI:10.1002/chir.22972

The separation methods of the enantiomers of two β-blockers and tryptophan were studied using capillary electrochromatography with heparin covalently as well as non-covalently, bonded onto the capillary inner wall as stationary phase and electrokinetic chromatography with heparin as pseudostationary phase. In the case of heparin, used as a stationary phase, the method was unable to resolve enantiomers in both cases β-blockers and tryptophan. On the other hand, when heparin was used as a pseudostationary phase, the resolution of the enantiomers was obtained only with 3-aminopropyltriethoxysilane which were immobilised onto the inner phase of the capillary. The results of this study let us infer that the electrostatic, hydrophobic, and steric interactions were involved in the separation mechanisms. The separation was achieved in less than 10?minutes under the optimized conditions: 30?mM phosphate buffer (pH?2.5) with the adding of 15?mg/mL of heparin at 15°C and 10?kV. The usefulness of heparin as a chiral selector both in electrokinetic chromatography using 3-aminopropyltriethoxysilane attached to the capillary was demonstrated for the first time. The developed method was powerful, sensitive, and fast, and it could be considered an important alternative to conventional methods used for chiral separation.

Full text of DOI:10.1002/chir.22972

Received: 27 October 2017
DOI: 10.1002/chir.22972
Revised: 14 March 2018
Accepted: 12 April 2018
R E G U L A R A R T I C L E
Enantiomeric separation of β‐blockers and tryptophan using
heparin as stationary and pseudostationary phases in
capillary electrophoresis
Yi Liu1 | Lorena L. Sombra2 | Patricia W. Stege3,4
1 Department of Pharmacy, Peking
Abstract
University People's Hospital, Beijing,
The separation methods of the enantiomers of two β‐blockers and tryptophan
China
2 INQUISAL, Department of Chemistry
National University of San Luis,
CONICET, Chacabuco y Pedernera, San
Luis, Argentina
3 INBIOMIS, Facultad de Ciencias Exactas
Químicas y Naturales, Posadas, Misiones,
Argentina
4 CONICET (National Council of Scientific
and Technological Investigations), Buenos
Aires, Argentina
were studied using capillary electrochromatography with heparin covalently
as well as non‐covalently, bonded onto the capillary inner wall as stationary
phase and electrokinetic chromatography with heparin as pseudostationary
phase. In the case of heparin, used as a stationary phase, the method was unable
to resolve enantiomers in both cases β‐blockers and tryptophan. On the other
hand, when heparin was used as a pseudostationary phase, the resolution of
the enantiomers was obtained only with 3‐aminopropyltriethoxysilane which
were immobilised onto the inner phase of the capillary. The results of this study
let us infer that the electrostatic, hydrophobic, and steric interactions were
involved in the separation mechanisms. The separation was achieved in less
than 10 minutes under the optimized conditions: 30 mM phosphate buffer
(pH 2.5) with the adding of 15 mg/mL of heparin at 15°C and 10 kV. The useful-
ness of heparin as a chiral selector both in electrokinetic chromatography using
3‐aminopropyltriethoxysilane attached to the capillary was demonstrated for the
first time. The developed method was powerful, sensitive, and fast, and it could
be considered an important alternative to conventional methods used for
chiral separation.
Correspondence
Patricia W. Stege, INBIOMIS, Facultad de
Ciencias Exactas Quimicas y Naturales,
Posadas, Misiones, Agentina.
Email: pwstege@gmail.com
Funding information
Consejo Nacional de Investigaciones
Científicas y Técnicas, Grant/Award
Number: CONICET PIP
11220150100768CO; Universidad Nacional
de San Luis, Grant/Award Number:
PROICO 2‐2816
KEYWORDS
APTES, capillary electrochromatography, chiral separation, electrokinetic chromatography, heparin
1 | INTRODUCTION
Currently, in the industry, the separation of chiral
drugs is dominated by high performance liquid chroma-
Many pharmaceutical compounds have one or more chiral
centers that are responsible for their optical activity. The
importance of chirality in the activity of these compounds
has long been recognized due to the fact that those
enantiomers exhibit different activities in human and ani-
mal bodies.1 Being able to define the amount of each in a
mixture has become of utmost importance for the pharma-
ceutical industry. Consequently, developments of methods
for enantiomer separation are extremely valuable.
tography.2-6 Gas chromatography and supercritical fluid
chromatography are other techniques that have a key
application in the separation of chiral drugs.7 However,
during the last decade, capillary electrophoresis (CE)
has received a great attention within this field due to its
high resolution, power, and selectivity.8,9
An important advantage of using CE to separate
chiral drugs is the flexibility that this technique presents
in adding, during the separation process, a variety of
Chirality. 2018;1–8.
wileyonlinelibrary.com/journal/chir
© 2018 Wiley Periodicals, Inc.
1

2
LIU ET AL.
compounds that could act as some chiral selectors like
cyclodextrins and its derivatives,10-12 macrolides,13,14 and
many other small molecules.15,16 In general, chiral
separations using CE can be achieved following two
strategies: the first one includes adding chiral additives
in the running buffer while the second one is based on
the modification of the capillaries inner wall with a
chiral selector.17-19
Among the compounds selected for these experi-
ments, β‐blockers were found, which is a group of drugs
used in various medical conditions especially in the
treatment of cardiac arrhythmias and cardio protection
after myocardial infarction.36 Furthermore, tryptophan
was used, an essential amino acid which has chiral char-
acteristics. The two β‐blockers used in this work, pindolol
and propranolol, are not selective agents, which means
that they have the same effect on both adrenergic
receptors β‐1 and β‐2. The amino acid is deeply involved
in the synthesis of serotonin, a hormone strongly related
with stress.
The goal of this work is to transfer the acquired
knowledge regarding the behaviour of heparin as a chiral
selector when it was used as a stationary and pseudo
stationary phase. Moreover, in this work, we have tried
to explain the role of 3‐aminopropyltriethoxysilane
(APTES) as a stationary phase when heparin is used as
an additive in the BGE.
Many researches have focused their attention in
macromolecules with chiral selection capacity such as
proteins20-22 and polysaccharides.23-26 Among them,
polysaccharides are one of the most promising chiral
selectors due to the fact that they have a great versatility
in the CE enantiomer separation. Many neutral polysac-
charides such as dextran, dextrin,27,28 laminaran,
pullulan,29 etc. as well as ionic polysaccharides such as
heparin,30,31 chondroitin sulphate,32 dextran sulphate,33
pentosan polysulphate,34 have been successfully employed
as chiral selectors in CE. The most important characteris-
tic of polysaccharides is the low absorbance they have in
UV spectra range which allows these substances to be
used in CE with UV detection. Thus, this is being consid-
ered as an important advantage to perform high detection
sensitivity. Another advantage of the charged polysaccha-
rides is that they can be used in high concentration in the
background electrolyte (BGE) due to their high solubility
in aqueous solutions. Moreover, because of the impor-
tance of polysaccharides in biological systems,35 the
studies of their stereo‐selectivity capacity have great
potential applications not only to develop new methods
of separation but also for future studies of selectivity
interaction mechanisms between polysaccharides and
different biomolecules or pharmaceutical products.
Heparin's chiral selective capacity in CE was
reported for the first time by Stalcup and Agyei in
1994.30 Heparin is a glycosaminoglycan with a molecu-
lar mass between 6000 and 30,000. It is a heterogeneous
mixture of variably sulphated polysaccharide chains
composed of α‐(1,4)‐linked D‐glucosamine and also L‐
iduronic or D‐glucuronic acids. These groups are
responsible for the strong anionic character, good solu-
bility, and electrophoretic mobility. The unit structure
of heparin is shown in Figure 1.
2 | MATERIALS AND METHODS
2.1 | Materials
Heparin (H4784), APTES, 1‐ethyl‐3‐(3‐dimethylamino-
propyl) carbodiimide hydrochloride (EDC, E1769) as well
as propranolol and pindolol were purchased from Sigma‐
Aldrich. D, L‐tryptophan was brought from the British
Drug Houses (UK). Dimethyl sulphoxide (DMSO) was
purchased from LabScan, Dublin, Ireland. Phosphoric
acid, sodium acetate, sodium hydroxide (1.0 M), and
hydrochloric acid (1.0 M) were obtained from Oy FF‐
Chemical Ab (Yli Ii, Finland); acetone was from
Mallinckrodt Baker (Deventer, The Netherlands). The
pH solutions (4, 7, and 10), used for calibrating the pH
meter, were purchased in Merck (Darmstadt, Germany).
2.2 | Instrumentation
The capillary electrophoretic experiments were carried
out with a Hewlett‐Packard 3DCE system (Agilent,
Waldbronn, Germany) equipped with a diode array detec-
tor (detection at 200, 214, and 254 nm) and an air‐cooling
FIGURE 1 Unit structure of heparin
employed for CE enantiomer separations

LIU ET AL.
3
device for the capillary cassette. Bare fused‐silica capil-
laries of 50 μm (I.D.) × 375 μm (O.D.) were purchased
from Optronis GmbH, Kehl, Germany. The length from
the capillary to the detector was of 30.0 cm with a total
length of 38.5 cm for all the experiments. A Lauda
Ecoline Re‐104 water bath (Lauda‐Königshofen,
Germany) was used to control the temperature of the
autosampler.
A Jenway 3030 pH meter (Jenway, Felsted, UK) and a
MeterLab PHM 220 pH meter (Radiometer, Copenhagen,
Denmark) were used in the pH measurements. Distilled
water was further purified with a Millipore water purifi-
cation system (Millipore S.A., Molsheim, France).
Millipore filters (Bedford, MA, USA) were used for filter-
ing BGEs.
acetone at 50 mbar for 2 hours. Then, the capillary was
flushed with acetone for 10 minutes, water for 5 minutes,
and phosphate buffer (pH 5.5 and IS20mM) for
60 minutes in order to eliminate the excess of unbounded
APTES.
APTES‐heparin covalently coated capillary
After preconditioning and coating the capillary with
APTES, the amino‐silanized capillary was treated with
heparin. Briefly, a heparin solution (3 mg/mL) dissolved
in 2‐mL phosphate buffer (pH 7.4 and IS = 20 mM) con-
taining 0.25 mg of EDC was passed through the capillary
for 1 hour. Finally, the capillary was rinsed with phos-
phate buffer (pH 7.4 and IS = 20 mM) to remove the
not immobilized heparin.
APTES‐heparin non‐covalently coated capillary
After being coated with APTES, the heparin non‐
covalently coated capillary was flushed with heparin
solution at 50 mbar for 40 minutes and left standing in
the coating solution for 60 minutes. Finally, the capillary
was rinsed with phosphate buffer at 15 minutes.
2.3 | Methods
2.3.1 | Sample and buffer preparation
Stock solutions of each racemic sample were prepared in
methanol (pindolol) or water (propranolol and trypto-
phan) at concentrations of 1.0 mg/mL. The sample
solutions for enantio‐separation were prepared by mixing
stock solutions and diluting it with water at concentra-
tions of 100 μg/mL. The stock solution of DMSO (1% v/
v) used as EOF marker was prepared in water.
2.3.3 | Calculation
The resolution between the studied analytes was deter-
mined by Equations (1) and (2).
The BGEs used were phosphate buffer, ionic
strength(IS) 20 to 40 mM, pH 2.5 and 3.5; also acetate
buffer IS 20 to 40 mM, pH 4.5 and 5.5. Both buffers were
prepared with their acids and sodium hydroxide. The pH
was adjusted to 1.0 M sodium hydroxide, 1.0 M hydro-
chloric acid, or acetic acid, respectively.
Rs ¼ 2ðt2 ‐ t1Þ=ðw1 þ w2Þ
(1)
where, Rs is resolution based on peak‐width; t1, t2 are
migration times of enantiomers; w1, w2 are baseline peak
widths of enantiomers; and
The solutions containing chiral selector were freshly
prepared by dissolving heparin in the BGEs. Before use,
the BGEs were passed through 0.2‐μm Millipore filters
using a Millipore vacuum system. All the solutions were
stored at +4°C.
Rh ¼ ðHi − HmÞ=Hi
(2)
where Rh is resolution based on peak‐height. If the base-
line separation of two peaks cannot be achieved, Rh is
applied. Hi is peak height of the lower peak of the two
peaks, and Hm is the height of the valley between the
two peaks.
2.3.2 | Coating procedures
Three types of immobilized capillaries were used: APTES‐
coated capillary, APTES‐heparin covalently, and non‐
covalent coated capillary.
3 | RESULTS AND DISCUSSION
3.1 | Results
APTES‐coated capillary
In a previous study, we developed a procedure for the
covalent and non‐covalent coating of heparin onto the
inner phase of the capillary. At that time, the coating
had the aim of studying the interaction between heparin
and lipoproteins.37 The presence of carboxylic groups all
along the heparin molecule allowed us to perform the
covalent interaction between the carboxylic group and
amino group from APTES, which is previously attached
Before coating, a new capillary was pre‐treated using the
following procedures: 15‐minute flush with 1 M NaOH,
15‐minute flush with deionized water, and 15‐minute
flush with acetone at a pressure of 940 mbar. After pre-
conditioning, APTES coating was attached to the inner
surface of the capillary as follows: the capillary was
flushed with 4% (v/v) APTES solution in anhydrous

4
LIU ET AL.
to the inner phase of the capillary. In covalent coating,
heparin was attached to APTES using EDC as a linker.
In this reaction, the terminal amine group of APTES
molecule reacted with the carboxylic group of heparin.
As a consequence, a stable amide bond was formed. On
the contrary, non‐covalent immobilization of heparin on
APTES‐coated capillary was based on ionic interaction
between the positively charges of anime groups of APTES
and the negatively charges of sulphate and/or carboxylic
groups of heparin. In the current work, heparin was used
as a chiral selector, and its function was evaluated as a
stationary and pseudostationary phase, to study the
enantio‐separations of two basic chiral β‐blockers,
propranolol and pindolol, and one basic aminoacid, tryp-
tophan (Figure 2).
3.2 | Capillary electrophoretic
measurements in APTES coating
With the purpose of studying the stability of the APTES
coating, the EOF movement was monitored using DMSO
marker by six successive runs at pHs 2.5, 3.5, 4.5, and 5.5
(Figure 3A) and at 15, 25, and 37°C (Figure 3B). Above
pH 5.5, the coating turns unstable, and the measurement
of the EOF was imprecise. The conditions selected to per-
form the following experiment were pH 2.5 and 15°C.
FIGURE 3 A, Behaviour of the μEOF related to different pHs.
Running conditions: 30 mM phosphate buffer; applied voltage,
10 kV; temperature, 15°C; injection mode injection 0.5 psi during
10 seconds, detection at 214 nm; B, Behaviour of the μEOF related
to different temperatures. Running conditions: 30 mM phosphate
buffer, pH 2.5; applied voltage, 10 kV; injection mode injection
0.5 psi during 10 seconds, detection at 214 nm
3.3 | Chiral separation in APTES‐heparin
coated capillary
Despite the stability of the covalent and non‐covalent
heparin, the coating of the capillary exceeded our expec-
tation. The separation of the chiral β‐blockers and trypto-
phan would have been impossible without the addition of
heparin in the BGE, even when heparin was present
attached to the capillary (Figure 4).
3.4 | Chiral separation in APTES‐coated
capillary with heparin as a pseudo
stationary phase
As it was mentioned earlier, this study includes the chiral
separation with the aggregation of heparin as a pseudo
stationary phase in a bared capillary and in an APTES
coated capillary. The concentration of the heparin added
to the BGE is essential to optimize the separation pro-
cess. Four concentrations were evaluated (5, 10, 15,
20 mg/mL). The separation was not successful with the
bared capillary. A better separation between the two
enantiomers (propranolol and pindolol) and tryptophan
was observed with an APTES‐coated capillary and the
addition of heparin to the BGE. In this sense, the amount
of heparin added to the BGE was evaluated in terms of
FIGURE
2
Chemical structures of drugs investigated. A,
Propranolol; B, pindolol; C, tryptophan

LIU ET AL.
5
FIGURE 4 Electropherograms of the unsuccessful chiral separation of A, propanolol (peak 1) and pindolol (peak 2) and B, tryptophan
using heparin as a stationary phase. Running conditions: 30 mM phosphate buffer, pH 2.5; applied voltage, 10 kV; temperature, 15 °C;
injection mode: hydrodynamic, injection 0.5 psi during 10 seconds, detection at 214 nm
resolution (Table 1). As it was a convenient choice, the
separations were performed with the addition of 15 mg/
mL of heparin in the BGE. The presence of the heparin
affects the electroosmotic flow by altering the ionic
strength as well as the conductivity and viscosity of the
buffer. Although the increase in ionic strength and vis-
cosity from the addition from heparin tends to decrease
the electroosmotic flow, this may be partly compensated
by the increased conductivity, hence, mobility of the
analytes. Also, further increases in heparin concentration
were limited by concomitant increases in current.38
Table 2 shows the results, in terms of resolution, of
the chiral separation under different conditions. The
best separation and the working conditions were phos-
phate buffer 30 mM, pH 2.5, temperature 15°C, and
voltage 10 kV with the addition of 15 mg/mL of hepa-
rin. The results of enantiomeric separations by CE with
15 mg/mL of heparin as a pseudo stationary phase are
shown in Figures 5 and 6.
3.5 | Evaluation of the separation process
In order to consider the reliability of the results obtained
during the chiral separation, we evaluated the repeatabil-
ity (n = 6 in the same capillary) and the reproducibility
(day‐to‐day) of the separation process. The results showed
that the average retention time (n
6.04 0.02 minutes (peak 1) and 6.09
(peak 2) for propanolol and 6.24
3) and 6.29
= 6) were
0.02 minutes
0.03 minutes (peak
0.03 minutes (peak 4) for pindolol with
an RSD lower than 0.4%. The reproducibility of the
separation was measured during three successive days
(n = 6), and the RSD was no more than 1.2%, confirming
that the results of the separation remained stable at least
for three days.
TABLE 1 Enantiomeric resolution with different conditions of
In aqueous solutions, heparin forms a helical struc-
ture in the same manner as in α‐(1,4)‐linked dextrins.31
It has been postulated that the presence of the sulphate
groups39 as well as the heparin helical superstructure40
both contribute to the enantiomeric separations. Bearing
in mind the quaternary structure adopted by heparin,
we could speculate that when heparin is added to BGE,
the sulphate groups are exposed in the solution and, thus,
are accessible to interact with chiral compounds. When
heparin is attached to the inner phase of the capillary,
heparin added to the BGE.
Heparin
Rs
concentration
(mg/mL)
Pindolol
Propranolol
Tryptophan
5
0.26
0.64
1.15
0.97
0.44
0.66
1.21
1.21
0.52
0.73
1.38
1.13
10
15
20

6
LIU ET AL.
TABLE 2 Enantiomeric resolution of propranolol and pindolol with different running conditions using heparin as a pseudo stationary
phase. Running conditions: a‐pH 2.5, applied voltage 10 kV, temperature 15 °C, Injection mode injection 0.5 psi during 10 s, detection at 214
nm. b‐ Running conditions: 30 mM phosphate buffer, applied voltage 10 kV, temperature 15 °C, Injection mode injection 0.5 psi during 10 s,
detection at 214 nm. c‐ Running conditions: 30 mM phosphate buffer, pH 2.5, temperature 15 °C, Injection mode injection 0.5 psi during 10 s,
detection at 214 nm.
aBuffer concentration (mmol/L)
bBuffer pH
cVoltage (kV)
Rs
20
30
40
50
5.5
4.5
3.5
2.5
20
15
10
Propranolol
Pindolol
0.13
0.15
1.15
1.21
0.50
0.70
0.44
0.67
‐
‐
‐
‐
0.43
0.64
1.15
1.21
0.24
0.55
0.77
0.75
1.15
1.21
FIGURE
5
Electropherogram of the chiral separation of
propranolol and pindolol using heparin as a pseudo stationary
phase in an APTES‐coated capillary. Running conditions: 30 mM
phosphate buffer, pH 2.5; applied voltage, 10 kV; temperature,
15°C; injection mode: hydrodynamic, injection 0.5 psi during
10 seconds, detection at 214 nm
FIGURE
6
Electropherogram of the chiral separation of
tryptophan using heparin as a pseudo stationary phase in an
APTES‐coated capillary. Running conditions: 30 mM phosphate
buffer, pH 2.5; applied voltage, 10 kV; temperature, 15°C; injection
mode: hydrodynamic, injection 0.5 psi during 10 seconds, detection
at 214 nm
the sulphate groups could stay hidden in the structure
although the carboxylic groups are the ones which take
part of covalent bonding with silanols of the capillary.
In this way, the access for the chiral compounds to the
sulphate groups became more delicate. Moreover, the
negative results in electrostatic interaction between hepa-
rin and APTES could be explained due to the fact that
both carboxylic and sulphate group take part in the inter-
action resulting in a few negatively charged groups avail-
able to interact with the chiral compounds. As a final
point, we can postulate that chiral selection capacity of
heparin could be based on the interaction of the chiral
compound, not only with the sulphate groups, but also
with the carboxylic groups. The moment when the car-
boxylic groups take part of the covalent interaction, hep-
arin loses its capacity as chiral selector.
Having in mind the explanation given above, it is log-
ical to suggest that the affinity of heparin is higher for the
tryptophan than the β‐blockers due to the exposition of

LIU ET AL.
7
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The present work presents a separation procedure in
which heparin are added to the BGE as a pseudo station-
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improved thanks to the presence of APTES attached to
the inner phase of the capillary. In general, stronger
interactions, as evidenced by higher resolution, were
observed between heparin and tryptophan. The method
described in this study are very promising in terms of
resolution and reproducibility. Further work including
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ACKNOWLEDGMENTS
This work was supported by Consejo Nacional de
Investigaciones Científicas y Técnicas (CONICET PIP
11220150100768CO) and Universidad Nacional de San
Luis (PROICO 2‐2816)
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