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

http://mail%20to:%[email protected]/

http://mail%20to:%[email protected]/



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

96 M.L. Pacheco et al. / Chemosphere 51 (2003) 95–108



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

M.L. Pacheco et al. / Chemosphere 51 (2003) 95–108 97



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

.




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 )


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


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

.




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 )


(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 )


(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

.




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

.




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 )


(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

.




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 )


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 )


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

.




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 )



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

.




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 Ã 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 Ã 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 )


(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

.




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.




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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