Acidos Humicos y Plaguicidas
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Transcript of Acidos Humicos y Plaguicidas
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Supramolecular interactions of humic acids
with organic and inorganic xenobiotics studied
by capillary electrophoresis
M.L. Pacheco a, E.M. Pe~nna-Meendez b,1, J. Havel a,*
a Department of Analytical Chemistry, Faculty of Science, Masaryk University, Kotl aar r sk aa 2, 611 37 Brno, Czech Republicb Department of Analytical Chemistry, Nutrition and Food Science, University La Laguna, Tenerife, Spain
Received 15 May 2002; received in revised form 4 October 2002; accepted 6 December 2002
Abstract
Methodology based on capillary electrophoresis (CE) to study humic acids (HAs)–xenobiotics interactions is pro-
posed. The interactions of HAs with organic and inorganic xenobiotics like paraquat, diquat, p,p0-DDE, p,p0-DDT,
potassium ferrocyanide, potassium ferricyanide, chloride, 4-nitrocatechol and other organic compounds were studied.
They were found to be of different kind depending on the structure of the xenobiotic molecule and on its charge
(neutral, positive or negative). Ion binding, hydrogen bonding, van der Waals forces, ligand exchange, hydrophobic and
hydrophilic adsorption, charge-transfer complexes and sequestration are some of the different mechanisms proposed to
bind inorganic and organic compounds to HAs.
It was also observed that some of the pollutants are strongly complexed (bound) only with some of the HA fractions
forming quite stable entities of supramolecular kind, which can migrate independently. In addition, the stability constantof HA–Cl (negatively charged species) was estimated to be log k ¼ 3:1 Æ 0:95. In order to explain the interaction between
negatively charged HAs and inorganic anions (like ClÀ, [Fe(CN)6]3À and [Fe(CN)6]4À), it is proposed that macropoly-
cyclic polyamine structures are present in HA supramolecules, and that they are responsible for such strong binding.
Ó 2003 Elsevier Science Ltd. All rights reserved.
Keywords: Pesticides; Pollutants; Complexes; Chloride; Cyanides; Macromolecules
1. Introduction
Humic acids (HAs) are organic macromolecules with
multiple properties and high structural complexity. Theyexist abundantly in soil, natural water and various ter-
restrial and aquatic environments. HAs behave as su-
pramolecules (Steed and Atwood, 2000) which are able
to polymerize, aggregate (Fetsch et al., 1998), form mi-
celles (Guetzloff and Rice, 1994) and might also form
supramolecular ensembles with other compounds.
For a long time the elucidation of the composition
and structure of HAs has been one of the most important
tasks of humus chemistry and many scientists have
worked on it (Kononova, 1966; Stevenson, 1981; Schul-ten, 1995). Nevertheless, recently Burdon (2001) pro-
posed that humic organic matter consists mainly of
mixture of plant and microbial constituents plus the
same constituents in various stages of decomposition (i.e.
plant/microbial mixtures of carbohydrates, proteins, lipids
and partially degraded lignins, tannins, melanins, etc.).
Regardless of the still unknown HAs structure and the
great efforts to elucidate, it is known that the major HAs
functional groups include carboxylic, phenolic, hydroxyl,
carbonyl, amine, amide and aliphatic moieties, among
others. Due to this polyfunctionality, HAs are one of the
most powerful chelating agents among natural organic
Chemosphere 51 (2003) 95–108
www.elsevier.com/locate/chemosphere
* Corresponding author. Tel.: +42-541-129-568; fax: +42-
541-211-214.
E-mail address: [email protected] (J. Havel).1 On leave from: Department of Analytical Chemistry, Nu-
trition and Food Science, University La Laguna, Tenerife, Spain.
0045-6535/03/$ - see front matter Ó 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0045-6535(02)00846-9
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substances. They are able to complex heavy metals
(Lubal et al., 1998; Kurk and Choppin, 2000), radio-
nuclides (Lubal et al., 2000; Pacheco and Havel, 2001),
inorganic anions (Leita et al., 2001), halogens (Lee et al.,
2001; Myneni, 2002), organic acids (Cozzolino et al.,
2001), aromatic compounds (Schulten et al., 2001; Nam
and Kim, 2002), pesticides and herbicides (Chien andBleam, 1997; De Paolis and Kukkonen, 1997; Schmitt
et al., 1997; Fang et al., 1998; Shigemasa and Mamoru,
1999; Gevao et al., 2000; Klaus et al., 2000), etc. These
studies show the importance of the interaction of HAs
with pollutants (which form complexes of different solu-
bility and chemical and biochemical stability) because
HAs can modify the uptake, bioavailability, transport,
fixation and toxicity of the xenobiotics in the environ-
ment. Various experimental techniques have been used to
study HAs and their interaction with pollutants like
NMR (Chien and Bleam, 1997; De Paolis and Kukko-
nen, 1997), mass spectrometry (MS) (Klaus et al., 2000;Kujawinski et al., 2002), circular dichroism (CD) (Shig-
emasa and Mamoru, 1999), fluorescence spectroscopy
(Fang et al., 1998; Piana and Zahir, 2000), infrared
spectrophotometry (IR) (Schnitzer and Kerndorff, 1980;
De Paolis and Kukkonen, 1997), differential thermal
analysis (DTA) (Schnitzer and Kerndorff, 1980), equi-
librium dialysis method (De Paolis and Kukkonen, 1997),
etc. Capillary electrophoresis (CE) has been found to be
one of the most efficient methods for characterization of
HAs (Norden and Dabek-Zlotorzynka, 1997; Fetsch and
Havel, 1998; Schmitt-Kopplin et al., 1998; Havel and
Fetsch, 2000; Pokornaa et al., 2000a,b; Havel et al., 2001;
Nagyova and Kaniansky, 2001; Pokornaa et al., 2001;
Hosse and Wilkinson, 2001). Due to its high sensitivity
and efficiency, some physicochemical HAs properties (i.e.
aggregation (Fetsch et al., 1998) and stability (Pokornaa
et al., 2001)) have been studied using this technique.
Despite the high number of studies done and re-
ported in the literature, still information concerning the
mechanisms of interaction between inorganic and or-
ganic pollutants with HAs needs to be clarified. There-
fore, the aim of this work is to apply CE technique to
enhance the understanding of the complex interactions
(bounds) between the HAs and various inorganic and
organic pollutants (xenobiotics) of environmental in-terest and propose some of the possible mechanisms of
their interaction.
2. Experimental
2.1. Chemicals
All reagents were of analytical grade purity. Isopro-
panol, sodium hydroxide, hydrochloric acid, sodium
chloride, 1,1,1-tris-hydroxymethyl-aminomethane (Tris),
ethylenediaminetetraacetic acid (EDTA), 3,3-[[1,1biphe-
nyl]4,4-diylbis(azo)-]bis[4-amino-1-naphthalenesulfonic
acid] disodium salt (Congo red), potassium ferrocyanide
and potassium ferricyanide were obtained from Lachema
(Brno, Czech Republic).
Methyl viologen dichloride hydrate (paraquat),
2,7-dibromo-5-[hydroxymercury]-fluorescein (Mercuro-chrome), 1,1-bis-(4-chlorophenyl)-2,2-dichloroethylene
( p,p0-DDE) and 1,1-bis-(4-chlorophenyl)-2,2,2-trichloro-
ethane ( p,p0-DDT) were purchased from Sigma-Aldrich
(Steinheim, Germany).
Boric acid was from Merck (Darmstadt, Germany).
Deiquat monohydrate (diquat) was from Riedel de
Haen (Munich, Germany). 4-Nitrocatechol was from
Loba-Chemie (Wien, Austria) and mesityloxide used as
an EOF marker was from Fluka (Buchs, Switzerland).
Deionized water used to prepare all solutions was
double-distilled from a quartz apparatus of Heraeus
Quartzschmelze (Hanau, Germany).The HA samples used in this work were Peat HA
standard (1S103H), Soil HA standard (1S102H) and
Leonardite HA standard (1S101H) from International
Humic Substances Society (IHSS) and coal-derived
Czech HA Chemapex standard sample from Chemapex
(Chomutov, Czech Republic).
2.2. Sample preparation
Humic acids: Stock solutions of 1000 mg lÀ1 of HA
were prepared by dissolving the corresponding weight in
36 mM NaOH. For CZE analysis the stock solutions
were diluted 10 times with water. All HAs working so-
lutions were analyzed after approximately 10 days of
preparation because it was found that after this time the
electropherograms are reproducible as described previ-
ously in Pokornaa et al. (2001).
DDE and DDT: Stock solution of 2.5 mM DDE was
prepared by dissolving 8 mg of the solid in 9 ml of iso-
propanol and 1 ml of water. The stock solution of 5 mM
DDT was prepared dissolving 9 mg of solid in 4 ml of
isopropanol and 1 ml of water.
HA–xenobiotic solution: HA solution (100 mglÀ1)
was mixed with proper amount of xenobiotic, shaken for1 min, injected and analyzed. All measurements of the
mixed solutions were done the same day.
2.3. Apparatus
Experiments were carried out on a Beckman (Model
P/ACE) System 5500 (Palo Alto, CA, USA) equipped
with a diode array detection (DAD) system, automatic
injector, fluid-cooled column cartridge and System Gold
Data station. Fused silica capillary tube of 37 cm (30.5
cm length to the detector) Â 75 lm I.D. was used. The
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normal polarity mode of the CZE system (cathodic pole
at the side of detection) was applied.
The pH was measured using a combined glass OP-
08080 electrode and Precision Digital pH-meter OP-208/
1 both of Radelkis (Budapest, Hungary) while standard
buffer solutions of Radiometer were used for the cali-
bration.
2.4. Procedure
All sample solutions were filtered using filters of 0.2
lm pore size of Sigma Co. (USA) before been injected
and analyzed. Fused silica capillary tube was condi-
tioned for the first time under the next procedure: 10 min
1 M NaOH at 40 °C, 10 min 0.1 M NaOH at 40 °C,
Table 1
List of compounds studied in this work
Compound class Name Structural formula
Organochlorine
pesticides
p,p0-DDT
p,p0-DDE
Quaternary-ammonium
pesticides ‘‘Quats’’Paraquat
Diquat
Pesticide metabolite 4-Nitrocatechol
Organic antibacterial Mercurochrome
Cyanide complexes Hexacyano iron(II) [Fe(CN)6]4À
Hexacyano iron(III) [Fe(CN)6]3À
Azo-dye Congo red
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10 min water at 30 °C and finally 10 min BGE at 25 °C.
The capillary was washed daily with deionized water for
10 min and 5 min with buffer solution. Between analysis
the capillary was washed for 3 min with deionized water
and for 3 min with BGE. Optimal separation conditions
used in this study were separation voltage of 20 kV, 20
°C temperature and 20 s hydrodynamic injection of samples. Detection wavelength was varied depending on
the sample measured. EOF was determined using 0.1%
mesityl oxide under the same conditions of the HAs
separation. At the end of the working day, the capillary
was washed for 1 min with 0.1 M NaOH, for 5 min with
deionized water and for 3 min with BGE.
3. Results and discussion
Based on a previous work (Pacheco and Havel, 2002)
a buffer consisting of 90 mM boric acid, 115 mM Tris
and 0.75 mM EDTA (pH 8.4) was used here as it was
found as an optimal one to obtain well reproducible
electropherograms. We have reported also that the
presence of sodium is strongly interfering the separation
patterns of HA (Pacheco and Havel, 2002). Therefore,
this buffer has been selected also because it is sodium
free. For all the study, Chemapex standard HA was
chosen because it is well characterized and studied
(Pokornaa et al., 2000a,b), additional experiments with
IHSS humic acids were also done.
All the xenobiotics studied in this work are presented
in Table 1.Methodology of capillary electrophoresis for HA–
xenobiotics affinity studies: In order to study affinity
of xenobiotics with HA, either (i) the mixture of HA +
xenobiotic was prepared and small volume was injected
into the capillary and analyzed, or (ii) studied com-
pound (xenobiotic) was added to the background elec-
trolyte at several concentration levels and pure solution
of HA was injected. In both cases, we were following the
changes in electrophoretic mobilities of HA or xenobiotic
peaks by applying high voltage.
3.1. Interaction of HA with non-ionic compounds
3.1.1. DDT and DDE
Two chlorinated pesticides, p,p0-DDT and p,p0-DDE
were selected as non-ionic pollutants because of their
environmental significance.
2 4 6 8 10 12
0.00
0.02
0.04
0.06
0.08
0.10
0.12
A b s o r b a n c e ( A U )
Migration time (min)
(n)
(m)
(l)
(k)
(j)
(i)
(h)
(g)
(f)
(e)
(d)
(c)
(b)
(a)
EOF
Fig. 1. Electropherograms for HA and different DDE additions: (a) 0.0, (b) 0.02, (c) 0.04, (d) 0.06, (e) 0.08, (f) 0.10, (g) 0.12, (h) 0.14,
(i) 0.16, (j) 0.18, (k) 0.20, (l) 0.30, (m) 0.40 and (n) 0.5 mM. Detection at k ¼ 257 nm. Concentration of HA: 100 mg lÀ1
.
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Because of limited solubility of DDE and DDT in
water the compounds were dissolved in isopropanol. A
solution of 100 mg lÀ1 HA was spiked with increased
amounts of either DDT or DDE, the mixture was in-
jected to the capillary and then analyzed (frontal anal-
ysis capillary electrophoresis). Fig. 1 shows changes in
the electropherogram when increasing additions of DDEwere applied. It is possible to observe that the main
group of peaks (initially between 3 and 4 min appro-
ximately) and the fraction with migration time appro-
ximately 11.5 min, in general are shifted to lower
migration time values. The same results were obtained
for DDT additions (Fig. 2(a) and (b)). However, per-
forming experiments with pure isopropanol leads to
similar patterns as those in Fig. 2 while the differences
were too small to follow the effect of DDE and DDT in
HA solutions. Isopropanol is decreasing the negative
mobility of the humics, most probably is encapsulatedby humics and increasing their molecular weight. In
conclusion, isopropanol is causing most of the changes
and from this experiment no conclusions can be drawn
as for the interaction of DDT and DDE with humics.
1 2 3 4 5 6
0.00
0.02
0.04
0.06
A b s o r b a n c e
( A U )
Migration time (min)
1 2 3 4 5 6
0.00
0.02
0.04
0.06
HA + DDE
HA
DDE
(b)
(a)
HA + DDT
HA
DDT
EOF
EOF
Migration time (min)
A b s o r b a n
c e ( A U )
Fig. 2. Fingerprints concerning various additions of neutral pesticides to HA solution: (a) 0.16 mM DDE and (b) 0.16 mM DDT.
Detection at k ¼ 257 nm. Concentration of HA: 100 mg lÀ1
.
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3.2. Interaction of HA with ionic compounds
Numerous studies have been done concerning the
study of interaction between macrocyclic ligands and
metal cations, nevertheless, just recently in a previous
work (Pacheco and Havel, 2002) the interaction of HA
with sodium cation was demonstrated. It was found,surprisingly, that the interaction Naþ –HA is higher if
sodium cation is not free but complexed with a crown
ether.
3.2.1. Cations
3.2.1.1. Paraquat and diquat. As examples of cationic
pesticides the paraquat and diquat were chosen in this
work. In order to study the binding of paraquat to HA,
batch experiments were done where paraquat was
added to HA. Fig. 3(a) displays the electropherograms
obtained for HA, paraquat and HA–paraquat solutions.
From the electropherograms obtained it follows that
addition of paraquat to HA results in a peak broadening
of the paraquat (migration time 1.3 min), the migrationtime of the ‘‘hump’’ constituted by the main group of
HAs peaks is higher and the resolution of these peaks
slightly increases. Similar results were obtained for ad-
dition of diquat to HA solution (Fig. 3(b)). We assume
that paraquat and diquat bind to HA supramolecules by
ionic binding resulting in charge neutralization. Also, it is
known that paraquat and diquat may be able to form a
0.00
0.02
0.04
0.06
0.08
A b s o r b a n c e
( A U )
Migration time (min)
(a)
HA + PARAQUAT
HA
PARAQUAT
1 2 3 4 5 6 7 8
0.00
0.02
0.04
0.06
A b s o r b a n c e
( A
U )
Migration time (min)
(b)
HA + DIQUAT
HA
DIQUAT
EOF
EOF
1 2 3 4 5 6 7 8
Fig. 3. Comparison of the electropherograms for HA solution and that one after addition of positively charged pesticide: (a) 0.7 mM
paraquat and detection at k ¼ 257 nm and (b) 0.7 mM diquat and detection at k ¼ 320 nm. Concentration of HA: 100 mg lÀ1
.
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charge-transfer complex with HAs (Gevao et al., 2000).
This means that the broader paraquat peak after the
addition of HA could be assigned to the formation of
charge-transfer complexes with quinone and diphenol
organic groups present in the HA structure, with electron-
deficient moiety and electron-rich centres, respectively.
Concerning the shift to high migration time of the
HA main group of peaks, the presence of several nega-
tively charged groups in HAs (carboxylic, phenolic,
hydroxyl, etc.) and positively charged paraquat results
in charge neutralization. However, chloride and/or
bromide ions (coming from the paraquat salt) might also
Fig. 4. 3D electropherograms concerning the addition of paraquat to HA solution: (a) 0.7 mM paraquat and (b) HA + 0.7 mM
paraquat. Concentration of HA: 100 mg lÀ1
.
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interact with the HAs and the final negative charge
predominates in the whole HA–anion macromolecule,
and as a consequence, the migration time of the ‘‘hump’’
increases. Interaction between HAs and negatively
charged compounds is described in the next section.
3D electropherograms of paraquat and HA–para-
quat mixture are shown in Fig. 4. The HA–paraquatinteraction is reflected in the lowering of the paraquat
peak height after the addition of HA.
3.2.2. Anions
Chemistry of the anions coordination with supra-
molecules has made progress in recent decades (Lehn,
1995). In this work a series of experiments were carried
out in order to study the interaction between HAs and
some anions like chloride, ferro- and ferricyanides, nitro-
and azo-compounds were chosen because of their envi-
ronmental implication. Concerning studied xenobiotic
compounds with exception of nitrocatechol, Mercuro-
chrome and Congo red, we have found no references
concerning the interaction with buffer components (Tris,
EDTA, borate). Compounds containing –OH groups
(nitrocatechol, Mercurochrome and Congo) are known
to react only with borate but this reaction in alkaline
region (pH 8.4) is diminished.
3.2.2.1. Chloride. Due to the interaction of sodium ca-
tions with HAs, a solution of Tris-chloride was prepared
from hydrochloric acid and Tris. In this case, because we
were interested in estimation of the approximate value
of the binding constant HA–Cl associate and due to the
low absorbance of the Tris-chloride solution, the capil-
lary was filled with the same buffer as always (90 mM
boric acid, 115 mM Tris and 0.75 mM EDTA) con-
taining in addition tris-chloride at various concentra-
tions, and then HA was injected and analyzed. This
mode of operation is known as affinity capillary elec-trophoresis (ACE). Fig. 5 shows the electropherograms
obtained. The shift of the peaks in electropherograms
to higher migration time values confirms that the
HA–Cl supramolecule becomes more and more nega-
tively charged while the main group of peaks becomes
broader, maybe due to the HA self-assembling after the
interaction. From the electropherograms shown in Fig. 5
the experimental electrophoretic mobilities (of the high-
est peak) were calculated according to Eq. (1),
leff ¼Lt Ld
V
1
t m
À1
t eof ð1Þ
where Lt is the total capillary length, Ld is the length of
the capillary from the injection side to the detector, t m
is the migration time, t eof if the migration time of the
neutral EOF marker, and V is the applied voltage. Using
the SQUAD program (Leggett and McBryde, 1975;
Meloun et al., 1988) the stability constant of the HA–Cl
assembly was estimated to be 3:1 Æ 0:95. Calculations
were also done with CELET program (Havel and Janoss,
1997) and the value obtained was in good agreement
with SQUAD program. This value reveals that the HA–
chloride interaction is rather strong and should not be
0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
A b s o r b
a n c e
( A U )
Migration time (min)
(f)
(e)
(d)
(c)
(b)
(a)
EOF
5 10
Fig. 5. Electropherograms concerning constant HA concentration and increasing levels of chloride in the buffer: (a) 0.0, (b) 0.4, (c) 0.7,
(d) 1.2, (e) 2 and (f) 5 mM chloride. Detection at k ¼ 210 nm. Concentration of HA: 100 mg lÀ1
.
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overlooked because of its environmental implications
(i.e. persistence of chloride in the soil, formation of
harmful organochlorine compounds, etc.). In soils, even
if chloride anion can be complexed by metal cations,
recently Myneni (2002) described that organo-Cl com-
pounds are the dominant forms of chloride in the or-
ganic fraction of soils, sediments and aquatic systems.In addition, positively charged (i.e. ammonium,
guanidinium moieties) or neutral electron-deficient
groups present in HA supramolecules, also may serve as
interaction sites for anion binding depending on elec-
trostatic and structural effects. Shulten and Schnitzer
(1998) identified high variety of nitrogen compounds in
HAs and soils which can be present inside the HA su-
pramolecule, forming macrocycles, macropolycycles,
structures similar to crown ethers or cryptands, etc., that
can interact strongly with anions like chloride. Some
macropolycyclic polyamines have already been reported
to bind chloride specifically (Lehn, 1995).
Therefore, we suggest that chloride might be bound
to macrocyclic polyamides or other similar structures in
humics. However, confirmation about the nature of
chloride binding site requires further study.
3.2.2.2. Ferricyanide and ferrocyanide. Soils close to in-
dustrial plants such as metallurgical or mining (amongothers) might contain large amounts of iron-cyanide
complexes. Therefore, because of the environmental
importance, in this work we have studied ferro- and
ferri-hexacyanides interactions with HAs. First of all,
redox reactions of HAs with cyanides were observed.
Fig. 6(a) shows the effect of HA addition to [Fe(CN)6]3À
solution (positive pole at the detector side). On
increasing HA concentration, the ferricyanide peak
(migration time 4.2 min) decreases and ferrocyanide
peak (migration time 5.6 min) is formed. This is the
evidence of Fe(III) reduction to Fe(II)-cyanide caused
by HAs.
1 2 3 4 5 6 7 8
0.000
0.005
0.010
0.015
0.020
0.025
A b
s o r b a n c e
( A U )
Migration time (min)
Fe(CN)64-
Fe(CN)63- + 800 mg/l HA
Fe(CN)63- + 500 mg/l HA
Fe(CN)63- + 225 mg/l HA
Fe(CN)63- + 100 mg/l HA
Fe(CN)6
3-
+ 50 mg/l HA
Fe(CN)63-
(a)
0 2 4 6 8 10
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
A b s o r b a n c e
( A
U )
Migration time (min)
HA + 15 mM Fe(CN)64-
HA + 5 mM Fe(CN)64-
HA + 1 mM Fe(CN)64-
HA
(b)
EOF
Fig. 6. Electropherograms concerning (a) additions of HA to constant ½FeðCNÞ63À ¼ 0:1 mM; reversed polarity and detection at
k ¼ 210 nm (½FeðCNÞ64À ¼ 0:01 mM) and (b) additions of [Fe(CN)6]4À to constant HA solution. Normal polarity and detection at
k ¼ 210 nm. Concentration of HA: 100 mg lÀ1
.
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Fig. 6(b) shows the electropherograms obtained after
addition of potassium ferrocyanide to Chemapex stan-
dard HA solution. On adding increasing amounts of
ferro-hexacyanide, changes in both the mobilities of the
HA fractions and the number of the peaks were ob-
served (resolution is lost). The same effect was observed
for ferricyanide. The shift to lower migration timevalues, which is not in correspondence with ferro- or
ferricyanide binding, can be explained assuming in-
crease of the HA–[Fe(CN)6]3À and HA–[Fe(CN)6]4À
supramolecules molecular weight in comparison to
original molecule. Leita et al. (2001) reported that both
HA–iron-cyanide complexes are much more stable than
the relative iron-cyanide complexes. The stability con-
stants of the HA–iron-cyanide complexes were found
around 1030 –1040 (Leita et al., 2001). It means that the
HA–iron-cyanide complexes are so stable that the
competition equilibrium of HAs with potassium and
sodium cations can, in this case, be neglected.
The conclusions concerning interaction of HA with
iron-cyanide complexes mentioned above, are also sup-
ported by Peter and Gross (1983), who studied themacrocyclic polyammonium receptor molecules which
are also able to complex specifically [Fe(CN)6]3À and
[Fe(CN)6]4À, yielding second-coordination-sphere com-
plexes known as ‘‘supercomplexes’’.
3.2.2.3. 4-Nitrocatechol . 4-Nitrocatechol is a known
pesticide metabolite. The results obtained for additions
of 4-nitrocatechol to HA solution are given in Fig. 7(a).
2 4 6 8 10
0.00
0.02
0.04
0.06
0.08
A b s o r b a n c e
( A U )
A b s o r b a n c e
( A U )
Migration time (min)
Migration time (min)
2 4 6 8 10
0.00
0.02
0.04
0.06
0.08
(a)
(b)*
4-NITROCATECHOL
HA + 4-NITROCATECHOL
HA
HA
MERCUROCHROME
HA + MERCUROCHROME
EOF
EOF
Fig. 7. Electropherograms concerning additions of negatively charged organic molecules to a solution of constant HA concentration:
(a) 0.1 mM 4-nitrocatechol and (b) 0.16 mM Mercurochrome. Detection at 210 nm. Concentration of HA: 100 mg lÀ1
.
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They reveal a strong interference in the HA fingerprint.
The loss of resolution in the main HA group of peaks
and the increase in the intensity of the nitrocatechol
peak are evidence of the HA–4-nitrocatechol species
formation. In the same time, the increment of the mo-
lecular weight of the HA–4-nitrocatechol causes the shift
of the peaks to lower migration time values. Lehn (1995)described how the guanidinium group as well as amidi-
nium group (which are present in HAs supramolecules)
serves as interaction sites for the binding of carboxylates
(phosphates and related species) since they may form
two chelating H-bonds. We suggest here that this might
also be a possible mechanism of interaction between 4-
nitrocatechol and HA macromolecules forming HA–
4-nitrocatechol supramolecules.
3.2.2.4. Mercurochrome. The possible interaction be-
tween HAs and relatively big negatively charged organic
molecules was also studied. As an example Mercuro-chrome divalent anion (sodium salt) was chosen. The
compound belongs to the fluorescein family and it is
used as an antibacterial agent. Fig. 7(b) shows the
change of the electropherograms after addition of Mer-
curochrome. Similar to the examples mentioned above,
the main group of peaks of HA is shifted and becomes
more narrow. Additionally, an increase in the signal
corresponding to the main peak of Mercurochrome
(marked as à in Fig. 7(b)) was observed. It is interesting
to note that also such rather big (complex) molecule
interacts with some binding sites of HAs. The shift of the
main group of peaks can be again explained via the in-
crease of the molecular weight of the HA–xenobiotic
(because of the addition of the contaminant to the
HA supramolecule). The interaction between carboxy-
late groups of Mercurochrome and ammonium sites
present in HAs (i.e. COOÀ –NHþ4 interaction (De Rob-
ertis et al., 1985)) is the possible mechanism of interac-
tion.
On the other hand, the presence of sodium ions insolution (coming from the salt) and their interaction
with the negatively charged groups of HAs should not
be overlooked in this case. Both mechanisms can take
place simultaneously.
3.2.2.5. Congo red . As an example of another organic
anion, we have chosen highly polar sulfonated azo-dye
(Table 1) to study its interaction with HAs. In this case
also main group of peaks is shifted towards migration
time corresponding to electroosmotic flow (Fig. 8). It is
possible to observe interaction between the azo-dye and
one peak marked as à in Fig. 8. This fact suggests that inthis case, the interaction between dye and HA fraction is
selective, maybe due to the interaction between sulfo-
groups (of the dye) and amino groups present in HAs, or
via the encapsulation of the sulfonated azo-dye inside
the specific possible ellipsoidal cryptates that can be
present in HAs.
3D electropherograms for HA, Congo red and mix-
ture of HA–Congo red are shown in Fig. 9. The
formation of stable HA–Congo red complex is re-
flected in the intensity of the signals observed in
Fig. 9(c) which are 10Â higher than in the spectra of
Congo red alone (Fig. 9(b)). Formation of supramo-
lecular ensemble HA–Congo red is evidenced because
2 4 6 8 10 12
0.00
0.05
0.10
0.15
0.20
A b s o r b a n c e (
A U )
Migration time (min)
(d)
(c)
(b)
(a)
*
*
*
EOF
Fig. 8. Electropherograms concerning additions of Congo red dye to a solution of constant HA concentration: (a) 0.0, (b) 0.01, (c) 0.03
and (d) 0.09 mM Congo red. Detection at k ¼ 210 nm. Concentration of HA: 100 mg lÀ1
.
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HA 3D electropherogram (Fig. 9(a)) was substantially
modified when Congo red was added (Fig. 9(c)).
3.3. Comparison with HAs from IHSS
As final experiments, interaction of Congo red with
HAs from International Humic Substances Society
(IHSS) was studied (Fig. 10). The electropherogramsshow that the (a) peat, (b) soil and (c) leonardite HAs
have similar behaviour as observed for Chemapex
standard HA and additions of Congo red. Therefore, the
same binding sites of various HAs are suggested for HAs
of different origin and source.
4. Conclusions
CE has been demonstrated to be a suitable technique
to follow chemical interactions between HAs and vari-
ous compounds.
The zwitterionic character of HAs allows the inter-
action of anions with positively charged groups of
humics and vice versa. Organic cations like those of
pesticides (diquat and paraquat) form rather labile HA–
pesticide associates. In the same time, there is strong
interaction of HAs with pesticides counter-anions
(chloride or bromide), which was proved by an inde-
pendent study using Tris-chloride. The stability constant
of the HA–chloride associate or {HA–chloride} supra-molecular species was estimated for the first time. It is
rather high (log k % 3:1) and it means that chlorides are
‘‘complexed’’ quite strongly with HAs.
Strong interaction was also observed between HA
and iron-hexacyanide complexes. Fe(III)-hexacyanide is
reduced by HAs to Fe(II)-hexacyanide.
In case of sulfonated azo-dye the interaction is so
strong that an aggregate of a certain fraction of HA with
Congo red anion is migrating independently.
It was found that isopropanol also interacts with HA
and thus changes the separation patterns, most probably
because it is encapsulated by HAs.
Fig. 9. 3D electropherograms concerning addition of Congo red dye to a HA solution: (a) Chemapex HA, (b) 0.03 mM Congo red and
(c) HA + 0.03 mM Congo red. Concentration of HA: 100 mg lÀ1.
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In general, the interactions between HAs and xeno-
biotics (organic or inorganic) are very complex and of
various kind (complexation, aggregation, intercalation,
charge neutralization, redox, etc.) and mostly several
different sort of interactions might take place at the same
time.
Acknowledgements
This work is a part of a research program within a
grant of Ministry of Education and Youth of the Czech
Republic, No. J07/98: 143100011. M.L. Pacheco wishes
to thank to Consejo Nacional de Ciencia y Tecnologia
(CONACYT, Mexico) for the fellowship given for her
doctorate studies at Masaryk University.
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