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Retention of Acidic Aromatic Compounds in Ion Exclusion
Chromatographic Separations
Bronislaw K. Glóda; Giorgio Perezb
a
Polish Academy of Sciences Medical Research Centre, Warsaw, Poland b Istituto di Cromatografia del
CNR, Monterotondo Stazione, (RM), Italy
To cite this Article Glód, Bronislaw K. and Perez, Giorgio(1997) 'Retention of Acidic Aromatic Compounds in Ion
Exclusion Chromatographic Separations', Journal of Liquid Chromatography & Related Technologies, 20: 7, 1005 — 1013
To link to this Article: DOI: 10.1080/10826079708010954
URL: http://dx.doi.org/10.1080/10826079708010954
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J. LIQ. CHROM. & REL. TECHNOL., 20(7), 1005-1013 (1997)
RETENTION OF ACIDIC AROMATIC
COMPOUNDS IN ION EXCLUSION
CHROMATOGRAPHIC SEPARATIONS
Bronislaw K. GlM,' Giorgio Perez2,*
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1
Polish Academy of Sciences
Medical Research Centre
Dworkowa 3
00-784 Warsaw, Poland
'Istituto di Cromatografia del CNR
c.p. 10
00016 Monterotondo Stazione (RM), Italy
ABSTRACT
Aromatic acidic compounds have been separated by ion
exclusion chromatography. The theoretical equation, which
relates the retention volume of an eluted acid to its dissociation
constant, failed for aromatics, which show higher retention
volumes than predicted. An important role is played by
hydrophobic adsorption, which can be increased by addition of
ion interaction reagents to the mobile phase .
INTRODUCTION
Ion exclusion chromatography (IEC) is a widely applied technique to
separate ionic compounds, since the retention of a single compound depends on
the ratio of concentrations of its ionised and neutral forms.' The characteristic
feature of IEC technique is the electric charge of dissociated ion-exchange resin
functional groups which has the same sign of the electric charge of the analysed
1005
Copyright 0 1997 by Marcel Dekker. Inc
GLOD AND PEREZ
1006
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ionic compound. It follows that samples containing negatively charged ions. as
dissociated acidic compounds. are separated on cation exchange resins with
anionic functional groups. For this purpose a large ion exchange capacity
column is preferred. which can be obtained maximising the column dimensions
and the functional group concentration in the support, and using strong ionexchange resins.
When such a column is filled up with water. which is pumped through it
as a mobile phase. the water molecules build up hydration spheres around the
dissociated functional groups of the support. Water contained in the pores of
the support and in the hydration spheres is immobilised and it forms the
stationary phase, in which the resin functional group ions and the hydrogen
ions are dissolved. The retention mechanism of ion exclusion’ is based on the
following phenomenon. Neutral. uncharged molecules are allowed to penetrate
the resin, while similarly charged coions are repulsed owing to the presence of
dissociated functional groups immobilised in the stationary phase. The
hydrated resin network behaves like a semipermeable membrane between the
stationary phase and the mobile phase. With the exception of the covalently
bound fimctional groups and of the counterions because of the electroneutrality
requirement. all other species are freely exchanged through such a hypothetical
membrane, which is permeable to neutral molecules. As a consequence. strong
acids, which are completely dissociated and. therefore. electrostatically repulsed
are eluted. not separated. in the column dead volume (VM), which corresponds
to the volume of the mobile phase in the column. On the other hand.
undissociated compounds can completely penetrate the resin and are also not
separated being eluted in a volume correspondent to the sum of the inner and
the dead volumes of the column. where the inner column volume is just the
volume of its stationary phase. This behaviour makes the determination of the
inner and dead column volumes straightforward as it has been confirmed by
Tanaka et al.3 Only the acids of intermediate strength. with their dissociation
constants falling in the range 10.- - 10.’. can be separated by this technique.
Ion exclusion can seldom be considered as the sole retention mechanism
even on an ion-exclusion resin. Like, in other chromatographic techniques. it is
classified according to the primary mechanism of solute retention. This
primary mechanism In IEC is coulombic repulsion between solute and
dissociated groups of the resin. Besides ion exclusion. let us recall hydrophobic
adsorption on the resin network as in reversed phase chromatography, size
exclusion.435effect of functional group screening in the analysed sample.
normal phase retention. van der Wads, and polar interactions of the sample
compound with the support
’
ACIDIC AROMATIC COMPOUNDS
1007
The aim of the presented paper is to investigate how hydrophobic
adsorption influences the retention of aromatic acidic compounds in ion
exclusion chromatographic separations. To this purpose, the retention volumes
of aliphatic and aromatic acids are compared with a theoretical model.
Furthermore, the addition of ion interaction reagents is studied in order to
investigate the possibility to govern hydrophobic adsorption.
EXPERIMENTAL
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Materials
Analytical reagent-grade sulphuric acid (Carlo Erba, Milan. Italy)
dissolved in triply distilled water at the selected concentration was used as
mobile phase. Tetrabutylamonium bromides used as ion-interaction reagents
were Aldrich-Chemie (Steinheim, Germany) products. All other chemicals
were Research Grade products and were used without further purification.
Apparatus
The chromatographc analyses were carried out by Beckman equipment
consisting of a 110B Solvent Delivery Module, a 340 Organizer, and a 160
Absorbance Detector, and equipped with a HPX-72-0Bio-Rad (kchmond,
USA) column (300 x 7.8 mm I.D.,
hydroxide form strong ion exclusion column
packed with 11 pm particles of polystyrene-divinylbenzene copolymer,
crosslinking WO).
The retention times of the eluted compounds were measured
by a Hewlett Packard HP 3394A Integrator.
Procedure
Triply distilled water was passed through a Millipore (Bedford, USA)
Milli-Q water purification system, filtered through a Millipore 0.45 pm
membrane filter, and degassed in an ultrasonic bath, before the addition of
sulphuric acid. The column was equilibrated for at least 1 hour prior to being
used.
All of the analyses were camed out at ambient temperature. Each
sample was injected six times and the average value of its retention time
was taken.
GLOD AND PEREZ
1008
15
10
+ perchloric
5
W nitric
0 chloracetic
Oformic
0
0 acetic
PKa
0 methanol
+
-5
-10
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Vr [rnl]
Figure 1. Effect of pK, values on retention volumes, VR. Bic-Rad Aminex ionexclusion HPX-87H organic acid analysis column, 300 x 7.8 mm I.D., hydrogen form
8% crosslinked cation exchanger, 9 pm particle diameter. Mobile phase: 1 mN H2S04.
Solute acids separated by pure ion-exclusion effect.
RESULTS AND DISCUSSION
Many models have been used to describe the IEC mechanism. The
simplest one. whch assumes pure water as a mobile phase, equal inner and
dead volumes. and the support functional groups completely dissociated. leads
to simple equations.6 The distribution coefficient, K,+ can be obtained from the
chromatographic data:
where VR and Vs are the retention and the stationary phase volumes,
respectively.
The influence of the dissociation constant of an acid on its retention
volume is shown in Figure 1. The characteristic for the ionexclusion s-shape
dependence between the pK, and retention volume can be observed.
Strong acids, llke perchloric or nitric, are eluted at the dead column
volume (I(d = 0). Very weak acidic compounds are eluted at a distribution
coefficient equal one. Other acids (chloroacetic, formic. acetic) are eluted
between these boundaries, as contirmation of the theoretical prediction^.'.^
ACIDIC AROMATIC COMPOUNDS
1009
Table 1
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Observed Retention Volumes (mL) Against Expected Retention Volumes
from pKa Values
Acid
PKa
Vr obs.
Vr exp.
Benzoic
Salicylic
Anisic
Tannic
4.20
3.00
4.47
4.32
72.00
44.87
41.09
20.03
7.4 - 8.2
6.0 - 6.8
7.6-8.4
7.5 - 8.3
From the s-shape of Fig. 1. the retention of other acids can be calculated.
In Table 1 the found retention volumes of aromatic acids are compared with the
calculated values. It can be observed that these compounds are characterised by
much higher retention than expected on the basis of a pure ion-exclusion. It
means that their retention is governed by the mixed ion-exclusion hydrophobic adsorption mechanism.'
A theoretical model can be proposed.
The distribution coefficient (&) in the case of acidic solutes can be
written as:
where the subscripts M and S refer to the mobile and the stationary phase,
respectively.
Under the conditions that a strong acid is used as a buffer and that its
concentration is much hgher than that one of solute, Eq. (2) can be written as:
where cf and ch are the functional groups and the buffer concentrations,
respectively, and K, is the dissociation constant of the acid.
GLOD AND PEREZ
1010
Table 2
Compansion of Retention Volumes (mL) and pK,, Values of Some Isomers
of Substituted Benzoic Acids. Mobile Phase: 1mN H2S04
Column: Aminex HPX-87H
Acid
OrthoVR
p%
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Toluic
Nitrobenzoic
Phthalic
3.91
2.17
2.98
59
9.6
11.8
Parap&
4.36
3.44
3.51
MetaVR
PK
VR
61.6
4.27
3.49
3.54
103.
88.0
4.5
55.0
45.3
In an analytical chromatographic process we assume linear adsorption:
where KK consists of the two effects: the ion-exclusion and hydrophobic
adsorption of the neutral molecule.
In the absence of adsorption the concentrations of the neutral molecules
in the both phases are equal. Since in ion-exclusion columns the concentration
of the hnctional groups is generally much higher than that of the solute, the
second term of the numerator can be generally omitted.
From Table 1 it can be read out that the aromatic compounds are
characterised by a very h g h retention. This is probably due to the interaction
between their nelectron and the resin network. This effect was found even
stronger for aromatic bases’’4whose retention was a hundred times higher than
that expected from the pure ion-exclusion effect.
As reported in Table 2. substituted aromatic acids are eluted mainly in the
order of ortho-, para-, meta-. Even if they show different values of their
dssociation constants, such differences seem to be too small to explain the big
changes in their retention. An explanation can be found in their different
molecular volumes. which are related to their surface area. It can be observed
that their retention is inversely proportional to their density.
ACIDIC AROMATIC COMPOUNDS
1011
Table 3
Retention Volumes (mL) of Isomeric Nitrobenoic Acids in Different Mobile
Phases (Bio-Rad Aminex Ion-Exclusion HPX-87H Organic Acid Analysis
Column, 300 x 7.8 mm ID., Hydrogen Form 8% Cross-Linked
Cation Exchanger, 9 pm Particle Diameter)
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Mobile Phase
Isomer
pK,
p [g/mL]
Water
1mN HISO( 0.5mM TBABr
ortho
Para
Meta
2.17
3.44
3.49
1.575
1.55
1.494
5.05
9.6
5.14
9.3
55
88
8.33
49.05
74.19
As an example, m-nitrobenzoic acid is the last isomer eluted in the
different experimental conditions, reported in Table 3. The meta- isomer is
characterised by the smallest density value, 1.494 g/mL, in comparison with
para- (1.55 g/mL) and ortho- (1.575 g/mL) isomers.
Since hydrophobic adsorption seems to play an important role in ion
exclusion chromatographic separations, it seems useful to investigate how to
govern it.
Hydrophobic adsorption can be decreased by addition of an organic
modifier to the mobile phase. As a matter of fact.’ it has been shown that
cvclodextrine added to the mobile phase could also decrease retention.
On the other side, an increase of the hydrophobic adsorption can be
obtained by addition of an ion-interaction reagent (IIR) to the mobile phase. In
other chromatographic techniques,* it was found that the addition of small
concentration of IIR to the mobile phase in the absence of buffer not only
improved the retention, but led to more symmetrical peaks and avoided the
dependence of the solute retention on its concentration. Also in IEC, IIR can
be added to the mobile phase.
Since the electrostatic interactions between solutes and hnctional groups
are vexy quick, there should not be time wasted waiting for column
stabilisation. To confirm such a hypothesis, we changed the mobile phase from
water to 0.5 mM tetrabutylamonium bromide (TBABr). The equilibrium was
reached after few minutes. The same effect was found for the opposite
variation.
1012
GLOD AND PEREZ
75
50
I
-para
25
c
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0
-*
0.1
--c0.2
c
-- - -.
0.3
0.4
0.5
ImMI
Figure 2. Influence of the tetrabutylammonium bromide concentration on the
nitrobenzoic acids retention. Chromatographic conditions as in Fig. 1.
The influence of the IIR concentration on the retention for the
isomeric nitrobenzoic acids is shown in Fig. 2. It can be seen that the
addition of a small concentration of TBABr improves the separation of the
isomers.
CONCLUSIONS
Aromatic acidic compounds are characterised by higher retention than
predicted by a pure ion exclusion mechanism. We suggested that the reason
can be found in a hydrophobic adsorption on the resin network, which is
constituted mainly of styrene and divinylknzene copolymerised resins. It
means that, in this case, the retention is governed by the mixed ion-exclusion hydrophobic adsorption mechanism.
This adsorption can be increased by the addition of an ionic interaction
reagent to the mobile phase. Since, for tlus purpose, a small concentration
(0.01 mM) of ionic interaction reagent can be used in the absence of buffer, the
overall result is an improvement of solute retention without influencing the
detection q-stem.
ACIDIC AROMATIC COMPOUNDS
1013
ACKNOWLEDGEMENTS
This work was partly supported (B.K.G.) by the Scientific Research
Council grant KBN 2P303.042.02. Bronislaw K.Gl6d is indebted to CNRNATO for assisting with his travel to Italy.
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2. G. L. Zhao, L. N. Liu. Chromatographia, 32,453 (1991).
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3. K. Tanaka. T. Ishizuka,.H. Sunahara, J. Chromatogr., 174, 153 (1979).
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Received July 3 1 , 1996
Accepted September 30, 1996
Manuscript 423 1
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