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384
Chiang Mai J. Sci. 2010; 37(3)
Chiang Mai J. Sci. 2010; 37(3) : 384-396
www.science.cmu.ac.th/journal-science/josci.html
Contributed Paper
Retention Behavior of Aromatic Amines with Some
Ionic Liquids Mobile Phase in Semi-micro HPLC
Yuppadee Nusai*, Hitoshi Koizumi, Masaki Tachibana, Kazue Tani, and Nobutoshi Kiba
Department of Applied Chemistry, Faculty of Engineering, University of Yamanashi,
Yamanashi, 400-8510, Japan.
*Author for correspondence; e-mail: [email protected]
Received: 5 January 2010
Accepted: 3 May 2010
ABSTRACT
This research was aimed at investigating the retention behavior of aromatic amines
with some ionic liquid mobile phases in semi-micro HPLC to explain the unclear separation
mechanism. Aromatic amines (m-aminophenol, benzylamine, N,N-dimethylaniline,
p-aminobenzoic acid, p-aminophenol, aniline, p-toluidine and N-methylaniline) were examined.
The retention behavior of aromatic amines is greatly affected by the column equilibration
time, ionic liquid concentration, mobile phase pH and alkyl chain length on the imidazolium
cation of the ionic liquid. Semi-micro HPLC was used to increase separation power and
sensitivity, and in addition to save on the mobile phase of the expensive ionic liquid. The
separation mechanism involves ionic and hydrophobic interactions.
Keywords: Ionic liquid; mobile phase; semi-micro HPLC; aromatic amine.
1. INTRODUCTION
Ionic liquids (ILs) are a type of salts that
o
are liquid at low temperature (<100 C)[1].
ILs are normally composed of relatively large
organic cations and inorganic or organic anions.
They are polar solvents, environmentally
harmless, nonvolatile, and nonflammable.
Furthermore, it is possible to design solvents
for specific applications by varying the lengths
and branching of alkyl chains of the anionic
core and the cationic precursor [2]. As a result
of these ILs properties, ILs have been widely
used in various chemical fields such as liquidliquid extraction [3-5], capillary electrophoresis
(CE) [6-8]. ILs have been used increasingly to
replace organic solvents for increasing the
safety of workers in chemical laboratories and
to decrease pollution in the environment.
Recently, aqueous solutions of ILs have
been used as mobile phases in conventional
HPLC [2, 9-10]. However, the ionic liquids
price is still high. Semi-micro HPLC is an
interesting technique because a lower amount
of solvent and a lower volume of sample
injection are used (0.5 - 2 μL) and better
sensitivity is achieved than with conventional
HPLC. The use of the miniature analytical
column reduces the expensive ILs solutions
and simultaneously the waste liquid pollution
in the environment [11].
Because a few of eight aromatic amines
were coeluted, it was difficult to determine
the retention factor. Therefore, we divided the
Chiang Mai J. Sci. 2010; 37(3)
aromatic amines into two groups as follows.
The first group consisted of aniline (An),
N-methylaniline (N-MA), N, N-dimethylaniline
(N, N-DMA) and benzylamine (BA). The
second group consisted of p-aminobenzoic
385
acid (p-ABA), m-aminophenol (m-Aph),
p-toluidine (p-To) and p-aminophenol (p-Aph).
The chemical structure and pKa value of these
aromatic amines are shown in Figure 1.
Figure 1. The chemical structure of analytes. (a) aniline(An); pKa = 4.70, (b) N-methylaniline
(N-MA); pKa = 4.85, (c) N, N-dimethylaniline(N,N-DMA) ; pKa = 5.15, (d) benzylamine(BA);
pKa = 9.33, (e) p-aminobenzoic acid(p-ABA) ; pKa1 = 4.65; pKa2 = 4.80, (f) m-aminophenol
(m-Aph); pKa1 = 4.37; pKa2 = 9.815, (g) p-toluidine(p-To); pKa = 5.10 (h) p-aminophenol
(p-Aph); pKa1 = 5.48; pKa2 = 10.46.
1-Alkyl-3-methylimidazolium-type
ionic liquids were used, namely, 1-ethyl3-methylimidazolium tetrafluoroborate
([EMIm][BF4]), 1-butyl-3-methylimidazolium
tetrafluoroborate ([BMIm][BF4]) and 1-hexyl3-methylimidazolium tetrafluoroborate
([HMIm][BF4]). The chemical structure of
these ionic liquids is shown in Figure 2. 1-Alkyl3-methylimidazolium tetrafluoroborates
have a maximum absorbance value at about
216 nm.
Figure 2. The chemical structure of ionic liquids.
R = C2H5 = [EMIm][BF4]; λmax = 216 nm,
R = C4H9 = [BMIm][BF4]; λmax = 216 nm,
R = C6H13 = [HMIm][BF4]; λmax = 216 nm.
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Chiang Mai J. Sci. 2010; 37(3)
In this study, aqueous solutions of ILs
([EMIm][BF4], [BMIm][BF4] and [HMIm][BF4])
were applied as mobile phases in semi-micro
HPLC to increase separation power and
sensitivity, and in addition to save on the
expensive ILs. Furthermore, we investigated
the separation mechanism with aromatic
amines. Various factors such as column
equilibration time, ionic liquid concentration,
mobile phase pH and alkyl chain length on
the imidazolium cation of the ionic liquid were
studied. This study is the first report using
some ionic liquids mobile phases in semimicro HPLC.
EXPERIMENTAL
2.1 Apparatus and Semi-micro HPLC
Analysis
The semi-micro HPLC system was
composed of a LC-10ADVP Pump (Shimadzu,
Japan), a 7520 injector with a 0.5 μL sample
loop (Rheodyne, USA), a L-5025 column
oven (Hitachi, Japan), and an 875-UV/Vis
detector with a 1 μL cell volume (Jasco, Japan).
A Develosil guard column (ODS-HG-S;
5 μm silica particle size; 10 mm 1.5 mm i.d.)
and a Develosil ODS-T-3 column (end capping;
3 μm silica particle size; 100 mm 2 mm i.d.;
trifunctional (polymeric); 20% carbon content;
3.4 μmol/m2) were used (Nomura Chemicals,
Japan). The chromatograms were recorded
on a D-2500 Chromato-Integrator (Hitachi,
Japan). All chromatograms were obtained by
isocratic elution at 0.2 mL min-1 flow rate. A
sample injection volume was 0.5 μL. UV
detection at 254 nm was used in the whole
study. The retention factor (k) was calculated
using this formula.
2.
k=
(1)
where t R is the retention time of the
analyte and t0 is the retention time of the
unretained compound(NH4Cl). Five replicate
injections were made to determine the
retention time, and average values were used
to calculate the retention factor. All
o
experiments were performed at 25 C.
2.2 Reagents
An, N-MA, N, N-DMA, BA, p-ABA,
m-Aph, p-To and p-Aph were purchased from
Tokyu Kasei (Japan). [EMImBF 4 ] and
[BMImBF 4] were purchased from Merck
(Germany). [HMImBF4] was purchased from
Wako Pure Chemical (Japan).
2.3 Preparation of Mobile Phases and
Standard Solutions
2.3.1 Mobile phases preparation
The stock solutions of specified ionic
liquids concentrations were prepared by
dissolving individually the known amounts of
ionic liquids with deionized (DI) water. The
stock solutions were filtered through 0.45 μm
nylon membrane filters and were stocked in
o
a refrigerator at 4 C. The stock solutions were
prepared weekly to protect the bacteria’s
growth.
The working mobile phases were
prepared by diluting the stock solution to the
required concentrations with DI water, and
then they were adjusted to the required pH
values with 10% (v/v) hydrochloric acid or
0.1 M sodium hydroxide. They were freshly
prepared before use.
There was no buffer utilization because
10% (v/v) hydrochloric acid was used for
adjusting the mobile phase pH value. 0.1 M
sodium hydroxide was only used when the
excessively low mobile phase pH value was
adjusted.
2.3.2 Standard solution preparation
The stock standard solutions of 10 mM
analytes were prepared by dissolving
individually the known amount of analytes
with 0.2 - 1 mL 1 M hydrochloric acid, and
then adjusting them to the required volume
Chiang Mai J. Sci. 2010; 37(3)
with pH 3.0 hydrochloric acid aqueous
solutions. They were stocked in a refrigerator
o
at 4 C and were prepared monthly.
The pH 3.0 hydrochloric acid aqueous
solution was prepared by adding 10% (v/v)
hydrochloric acid to DI water. 10% (v/v)
hydrochloric acid was gradually added to DI
water until the aqueous solution pH value
equaled 3.0 by pH meter measure.
The 1 mM mixed working standard
solutions were prepared by diluting the 10
mM stock standard solutions with pH 3.0
hydrochloric acid aqueous solution. They were
divided into two groups. The first group
consisted of m-Aph, BA, N,N-DMA and
p-ABA ( 1 mM mixed aromatic amine series
1 standard solution). The second group
consisted of p-Aph, An, p-To and N-MA
(1 mM mixed aromatic amine series 2
standard solution). They were freshly prepared
daily to avoid potential errors from
decomposition of the compounds prior to
387
analysis with semi- micro HPLC.
3. RESULTS AND DISCUSSION
3.1 Effect of Column Equilibration Time
on the Retention Factor (k) of Aromatic
Amines
The column equilibration time is an
important factor in HPLC analysis. If the
column is not completely equilibrated, the
retention factor of analytes does not provide
a constant value. The experiment was
performed by using 5 mM (0.10% (w/ v) or
0.99 g L-1) [EMIm][BF4] aqueous solution at
pH 4.0 as the mobile phase, an ODS-T-3
column, and N-MA as a test sample. The
relationship between the retention factor of
N-MA and the equilibration time is shown in
Figure 3. It was found that when the column
equilibration time was gradually increased
from 1 to 18 hours, the N-MA retention
factor steadily decreased until stable at 15
hours. Accordingly, the column equilibration
Figure 3. Effect of the column equilibration time on the retention factor (k) of N-MA.
column: ODS-T-3(end capping; 3 μm; 2.0 mm i.d. 100 mm) ; chromatographic condition:
mobile phase: 5 mM [EMIm][BF4] aqueous solution at pH 4.0; flow rate: 0.2 mLmin-1; UV
detection: 254 nm; sample injection volume: 0.5 μL.
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Chiang Mai J. Sci. 2010; 37(3)
time of the ODS-T-3 column was selected at
15 hours throughout all experiments. The
aspect of [EMIm][BF 4] adsorbed on the
ODS-T-3 column is shown in Figure 4. We
can explain that gradually increasing the
column equilibration time makes ethyl and
methyl groups on imidazolium cations
gradually interact with octadecyl and methyl
groups of the stationary phase. The adsorbed
imidazolium cations layer is formed gradually
(pseudo-positive stationary phase); therefore,
the protonated N-MA molecule is gradually
eluted by electrostatic repulsion. After the
pseudo positive stationary phase is completely
formed, the retention factor of N-MA is
consequently constant.
Figure 4. The aspect of [EMIm][BF4] adsorbed on ODS-T-3 column.
3.2 Effect of [EMIm][BF4] Concentration
on the Retention Factor (k) of Aromatic
Amines
The preliminary study had shown that the
concentration of the ionic liquid affects the
retention behavior of the aromatic amines. A
series of 0.0, 1.0, 2.5 and 5.0 mM (0.00, 0.02,
0.05, 0.10% (w/ v) or 0.00, 0.20, 0.49, 0.99 g
L-1) [EMIm][BF4] aqueous solutions at pH 3.0
was examined as the mobile phase to separate
the 1mM mixed aromatic amine series 1,2
standard solutions. The result is shown in
Figure 5. It was found that when the aqueous
mobile phase without adding the [EMIm]
[BF4] at pH 3.0 (a1, a2) was used, there were
three problems as follows. First, band tailing
of all aromatic amines occurred. Second, pABA and N,N-DMA, p-To and N-MA could
not be separated. Third, the N,N-DMA peak
was very broad. On the other hand, when the
aqueous mobile phase containing 1 mM
(0.02 % (w/ v) or 0.20 g L-1) [EMIm][BF4] at
pH 3.0 (b1,b2) was used, it was found firstly
that band tailing of all aromatic amines was
greatly improved and symmetrical peaks of
all aromatic amines were obtained. This is due
to the fact that each ethyl and methyl group
of imidazolium cations interacts with the
octadecyl and methyl groups of the stationary
phase by hydrophobic interaction, respectively.
This is a disability of the alkyl groups of the
stationary phase, which leads to the decrease
in the possibility of dispersion interaction
between the analytes and alkyl groups of the
stationary phase. Furthermore, ionic liquids
can suppress residual silanols on the ODS
stationary phase even though end capping
type. Secondly, the N,N-DMA and p-ABA
peaks could be separated. Even when p-To
and N-MA peaks could not be separated, the
peak shape of these two analytes was sharper.
Thirdly, the retention time of N, N-DMA was
shorter than the retention time of p-ABA, and
the N, N-DMA peak was clearly more
symmetrical.
Chiang Mai J. Sci. 2010; 37(3)
389
Figure 5. Chromatograms of Ar-amines with aqueous mobile phases containing various
[EMIm][BF4] concentrations at pH 3.0. (a1,a2) 0 mM, (b1,b2) 1.0 mM, (c1,c2) 2.5 mM, (d1,d2)
5 mM. Chromatographic conditions: column: ODS-T-3(end capping; 3 μm; 2.0 mm i.d.
100 mm); flow rate: 0.2 mLmin-1; UV detection : 254 nm; sample injection volume: 0.5 μL.
Peaks : (1) m-Aph; (2) BA; (3) p-ABA; (4) N,N-DMA, (5) p-Aph, (6) An, (7) p-To and
(8) N-MA.
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Chiang Mai J. Sci. 2010; 37(3)
Figure 6A and 6B show the effect of
[EMIm][BF4] concentration in the aqueous
mobile phase on the retention factor (k) of
aromatic amines. We found that increasing the
[EMIm][BF 4] concentration affected the
retention factor of all aromatic amines. It
drastically decreased at 1.0 mM, and then
slightly decreased at 2.5 mM and 5 mM. We
can explain that the addition of 1.0 mM
[EMIm][BF4] to the aqueous mobile phase,
which causes the interaction between each ethyl
and methyl group on imidazolium cations and
octadecyl and methyl groups of the stationary
phase. The surface of the ODS stationary
phase is a positive charge; therefore, the
protonated aromatic amines are rapidly eluted.
It results in a decrease in the retention factor
of the aromatic amines. Afterwards, when the
concentration of [EMIm][BF4] was increased
A
to 2.50 mM, the retention factor of all
aromatic amines was constant because the
formation of the adsorbed imidazolium
cations layer (pseudo-positive stationary phase)
is complete. Moreover, when the concentration
of [EMIm][BF4] was further increased to 5
mM, the result was that the retention factor
of all aromatic amines slightly decreased.
We conclude that forming the adsorbed
imidazolium cations layer is equivalent above
2.5 mM [EMIm][BF4] concentrations.
Considering the pKa value order of
aromatic amines is as follows: BA>
p-Aph>N,N-DMA>p-To>N-MA>An>
p-ABA>m-Aph, the obtained retention
factor order from using 1.0, 2.5 and 5 mM
[EMIm][BF4] aqueous solutions at pH 3.0 as
mobile phase was p-Aph<m-Aph<An<BA
<p-To=N-MA<N,N-DMA<p-ABA. From
B
Figure 6. Effect of [EMIm][BF4] concentration in aqueous mobile phase at pH 3.0 on the
retention factor (k) of aromatic amines.
Chiang Mai J. Sci. 2010; 37(3)
the obtained retention factor of all aromatic
amines, we explain that each ethyl and methyl
group on imidazolium cations interacts with
octadecyl and methyl groups of the stationary
phase by hydrophobic interaction, respectively.
It makes the stationary phase surface become
the positive charge. When the aromatic amine
molecules are protonated, they become the
positive charge as well. Consequently, they
are eluted by electrostatic repulsion. If the
retention behavior mechanism is mainly
dominant by electrostatic repulsion, the
aromatic amine will be eluted in the order
of the pKa value. However, the obtained
retention factor of the aromatic amine was
not in accordance with the pKa value order.
This is because of the methyl group effect on
N-MA, N,N-DMA and p-To. From the
methyl group effect, there is important
evidence that indicates that the stationary
phase surface still has a hydrophobic part as
well. Accordingly, the separation mechanism
involves electrostatic repulsion and hydrophobic interaction.
391
3.3 Effect of Mobile Phase pH on the
Retention Factor (k) of Aromatic Amines
It is understood well that the retention
of the ionizable compounds on the reversed
phase column depends on the pH of the
aqueous portion in the mobile phase. For this
reason, 2.5 mM (0.05% (w/ v) or 0.49 g L-1)
[EMIm][BF4] aqueous solutions at various pH
values (3.0, 3.5 and 4.0) were examined as
mobile phases. The HPLC condition was
started at a pH value of 3.0 for the following
reasons: (1) The usable pH range of the
mobile phase for the ODS column using the
silica as the support material is 2-8. If a mobile
phase with a pH value below 2 is used, the
siloxanes linkage will crack. Moreover, a mobile
phase with a pH value more than 8 is used,
the silica will be dissolved. (2) All analytes can
be absolutely protonated to cation forms at a
pH value of 3.0.
Figure 7A and 7B show the effect of the
mobile phase pH on the retention factor (k)
of aromatic amines. It was found that when
the mobile phase pH was increased from 3.0
A
B
Figure 7. Effect of mobile phase pH on the retention factor (k) of aromatic amines.
392
to 3.5, the retention factor of An, N-MA, N,
N-DMA, m-Aph, p-To and p-ABA increased,
except BA and p-Aph. This behavior can be
explained as follows: these seven aromatic
amines are weak base, with the exception of
BA, which is strong base; consequently,
they are completely protonated to cation form
in acidic aqueous mobile phase (pH 3.0).
When the pH of aqueous mobile phase was
increased to pH 3.5, they can dissociate
less to cation form; however, most of the
aromatic amines are still cation form, with the
exception of p-ABA, which is a zwitterion.
It causes a decrease of electrostatic repulsion
force between the protonated aromatic
Chiang Mai J. Sci. 2010; 37(3)
amines and the positive stationary phase
surface. When the mobile phase pH was
further increased to 4.0, the retention factor
of An, N-MA, N,N-DMA, m-Aph and p-To
increased. It is because they can decreasingly
dissociate to cation form, which causes a
decrease of electrostatic repulsion force.
As for the retention factor of p-ABA, it
slightly decreased because it is zwitterion.
It was observed that the mobile phase pH
did not affect the retention factor of BA and
p-Aph, because both analytes are always
protonated species under the examined pH
value. The typical chromatograms are shown
in Figure 8.
Figure 8. Chromatograms of Ar-amines with aqueous mobile phases containing 2.5 mM
[EMIm][BF4] at various pH values (a1,a2) 3.0, (b1,b2) 3.5, (c1,c2) 4.0. Chromatographic
conditions were shown in Figure 5. Peaks: (1) m-Aph, (2) BA, (3) p-ABA, (4) N,N-DMA,
(5) p-Aph, (6) An, (7) p-To and (8) N-MA.
Chiang Mai J. Sci. 2010; 37(3)
3.4 Effect of Alkyl Chain Length on the
Imidazolium Cation of Ionic Liquid on
the Retention Factor (k) of Aromatic
Amines
Figure 9A and 9B show the effect of
alkyl chain length on the imidazolium ring of
ionic liquid on the retention factor (k) of
aromatic amines. This factor was investigated
by using 2.5 mM (0.05 % (w/ v) or 0.49g
L-1) [EMIm][BF4], 2.5 mM (0.06 % (w/ v) or
0.57 g L-1) [BMIm][BF4 ] and 2.5 mM(0.06 %
(w/ v) or 0.64 g L-1) [HMIm][BF4] aqueous
solutions at pH 3.0 as the mobile phases.
It was apparent that with the increase of
length of the alkyl substituent of imidazolium
cation from ethyl, butyl to hexyl, all retention
factors of aromatic amines decreased greatly.
The longer alkyl chain can interact with the
octadecyl more than the shorter alkyl chain; it
393
makes the stationary phase surface become
the positive charge more. It is the cause of
increasing the repulsive force of the same
positive charge between the stationary phase
surface and the ionized aromatic amines.
N,N-DMA is stronger base than N-MA.
If the separation mechanism of aromatic
amines is controlled by electrostatic repulsion,
N,N-DMA will be eluted more firstly than
N-MA. However, due to the fact that the
retention factor of N,N-DMA is longer
than N-MA, this separation mechanism has
an effect on account of hydrophobic force.
These indicated that increasing the alkyl
chain length on the imidazolium cation of
ionic liquid increases the hydrophobic force
as well. The typical chromatograms are
shown in Figure 10.
A
B
Figure 9. Effect of alkyl chain length of ionic liquid on the retention factor (k) of aromatic
amines.
394
Chiang Mai J. Sci. 2010; 37(3)
Figure 10. Chromatograms of Ar-amines with aqueous mobile phases containing 2.5 mM
various ionic liquids at pH 3.0. (a1,a2) [EMIm][BF 4], (b1,b2) [BMIm][BF 4], (c1,c2)
[HMIm][BF4]. Chromatographic conditions were shown in Figure 5. Peaks: (1) m-Aph,
(2) BA, (3) p-ABA, (4) N,N-DMA, (5) p-Aph, (6) An, (7) p-To and (8) N-MA.
Chiang Mai J. Sci. 2010; 37(3)
3.5 The Separation Mechanism with Ionic
Liquid Mobile Phase for Aromatic Amines
According to the obtained results of
retention behavior of aromatic amine;
therefore, we suppose that the separation
mechanism with ionic liquid mobile phase for
aromatic amines depends on repulsion
between the imidazolium cations which
interact with the methyl and octadecyl
groups of the octadecylsilica stationary phase
surface by hydrophobic interaction and the
protonated aromatic amines. When the
stationary phase surface becomes more
395
positive with ILs by the concentration, pH
and alkyl chain length, it causes the protonated
aromatic amines to be primarily repulsed by
ionic interaction. Furthermore, because of
other parts of imidazolium cations molecules
are still hydrophobic, the protonated N-MA
and N,N-DMA are also eluted by hydrophobic interaction. Consequently, the
separation mechanism involves ionic and
hydrophobic interactions. The separation
mechanism with ionic liquid mobile phase for
aromatic amines is shown in Figure 11.
Figure 11. The separation mechanism with ionic liquid mobile phase for aromatic amines.
4. CONCLUSION
The investigation indicated that the ionic
liquids are potential to solving problems of
aromatic amines separation such as band tailing
and band broadening, because ionic liquids
can play a multiplicity of roles, such as blocking
residual silanol groups, modifying the
stationary phase, or acting as ion-pairing
agents. Furthermore, increasing the ionic liquid
concentration and the alkyl chain length of
ionic liquid makes the retention factor of all
aromatic amines decrease clearly. On the
contrary, the increase of the mobile phase pH
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Chiang Mai J. Sci. 2010; 37(3)
causes the retention factor of most aromatic
amines to increase. The separation mechanism
depends on competition between the
imidazolium cations and the protonated
aromatic amines in interacting with the
stationary phase. The imidazolium cations
can better interact, and make the stationary
phase surface becomes the positive charge.
Therefore the protonated aromatic amines are
repulsed by ionic interaction. In addition,
because of another part of imidazolium cation
molecule is still hydrophobic, the protonated
aromatic amines are eluted by hydrophobic
interaction as well. These results are extremely
useful for applying this system to analyse other
compounds in the future.
[5] Carmichael A.J., Earle M.J., Holbrey J.D.,
McCor mac P.B. and Seddon K.R.,
The Heck reaction in ionic liquids: a
multiphasic catalyst system, Org.
Lett.,1999; 1: 997-1000.
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