Open Journal of Physical Chemistry, 2011, 1, 55-60
doi:10.4236/ojpc.2011.13008 Published Online November 2011 (http://www.SciRP.org/journal/ojpc)
Radical Stabilization in Dissolved Humates
Barbora Bakajová1, Ray von Wandruszka2*
1Faculty of Chemistry, Brno University of Technology, Brno, Czech Republic
2Department of Chemistry, University of Idaho, Moscow, ID, USA
*E-mail:
Received May 28, 2011; revised August 9, 2011; accepted Septe mbe r 12, 2011
Abstract
Quinoid entities, in which quinone and hydroquinone groups equilibrate via a semiquinone radical interme-
diate, are a common structural feature in humic materials. The electron paramagnetic resonance (EPR) sig-
nals of these radicals are significantly enhanced in the presence of diamagnetic divalent metal ions such as
Mg2+, while monovalent ions do not show the effect. The addition of trivalent ions leads to rapid precipita-
tion, leaving little room for observation. It was noted that the metal ions producing EPR signal enhancement
were also underwent effective bridging interactions with humic subunits, forming pseudomicellar structures.
Particle growth determined through dynamic light scattering measurements coincided with the onset of EPR
signal enhancement, and surface tension measurements further corroborated the coincidence of aggregation.
The addition of a chaotrope (urea), which broke up the humic structures, eliminated the EPR signal increases.
These observations strongly suggested that bridging interactions by divalent metal ions, and the intramo-
lecular aggregation that accompanied it, led to significant stabilization of semiquinone radicals within the
humic structure.
Keywords: Humic Acid, Semiquinone, EPR Enhancement, Bridging Interactions, Aggregation, Radical
Stabilization
1. Introduction
The transformation of natural organic matter leads to the
formation of humic substances. Under aerobic conditions
of composting or humification in terrestrial ecosystems
the main components of these materials are fulvic acid
(FA) and humic acid (HA). They are a complex, opera-
tionally defined suite of substances built around a highly
aromatic backbone and containing an abundance of oxy-
genated functionalities, including carboxylic, phenolic,
alcoholic and carbonyl groups [1].
Structural changes during ageing of HA lead to an in-
crease in the number of polyphenolic and quinoid units.
The latter generally exist as equilibrated quinone/
Hydroquinone structures encompassing a radical semi-
quinone intermediate that can be investigated by electron
paramagnetic resonance spectroscopy (EPR) [2].
The quinone-semiquinone-hydroquinone equilibria are
sensitive to various chemical and physical factors such as
redox conditions, radiation, pH, and metal concentration
[2,3]. Jerzykiewicz et al. [4] noted that the carboxylic,
phenolic and quinoid groups of HA are involved in metal
complexation, and interactions of humic acids with metal
ions can play a significant role in the shifting the reaction
equilibria. The effect of metal binding can be observed
as a change in the free radical structure and/or spin con-
centration [4-6].
The metal ions that affect the quinoid equilibria in
HAs also play a significant role in their aggregation beha-
vior [7]. Under the influence of the metals, the materials
aggregate to form pseudomicellar structures similar to
surfactant micelle, but constrained by the intramolecular
nature of the arrangement and the polydispersity of the
material [8,9]. Upon the addition of cations, especially
polyvalent ones, HA folds and shortens, forming compact
structures with relatively hydrophobic interiors and hy-
drophilic surfaces. This it thought to be due to a combina-
tion of charge neutralization and functional group bridg-
ing [10].
The present study was prompted by the observation
that the quinoid EPR resonance of HA is strongly enhan-
ced by the presence of metal ions, especially Mg2+ [11],
which also has been shown to have a major influence on
its aggregation behavior. The possible correlation be-

B. BAKAJOVÁ ET AL.
56
tween these two phenomena has been investigated.
2. Materials and Methods
2.1. Chemicals
The source of HA in this study was a South Moravian
lignite collected from the Mir mine in the area of Mikul-
cice, near Hodonin, Czech Republic. HA were extracted
by standard alkaline method with 0.5 M NaOH and 0.1
M Na4P2O7. After separation from parental lignite, humic
acids were precipitated with 4 M HCl, demineralised
with 0.5% HF, dialyzed against distilled water until chl-
oride and fluoride free, and freeze-dried.
Sodium chloride (Fisher Scientific), lithium chloride
(Allied Chemical, NY), magnesium chloride hexahydrate
(Baker), barium chloride (Fisher Scientific), calcium
chloride (EM Science), zinc chloride (Fisher Scientific),
aluminium chloride hexahydrates (Fisher Scientific), sa-
marium chloride (Aldrich), cerium chloride (Fisher Sci-
entific), ethylene-bis(trimethylammonium) iodide (Aldri-
ch), potassium bromide (Fisher Scientific), sodium hydr-
oxide (Fisher Scientific), hydrochloric acid (EMD Che-
micals), 3-carbamoyl-2,2,5,5,-tetramethyl-3-pyrrolin-1-
yloxy free radical 97% (Acros), and urea (Baker) were
used without further purification.
2.2. Procedures
For EPR analysis HA solutions (500 mg/L) were pre-
pared by dissolution in water with minimal addition of
NaOH. Various metals were added as their chlorides in
the concentration ranges 5.0 × 10–5 - 1.0 × 10–3 M. The
cations in question were Na+, Li+, Mg2+, Ba2+, Ca2+, Zn2+,
Al3+, Sm3+ and Ce3+; one non-metallic cation, the ethylene-
bis(trimethylammonium) ion [CH33N-CH22-NCH33]2+
(“N2”) at a concentration of 1.0 × 10–3 M, was also in-
cluded.
EPR spectra were recorded on a Bruker EMX 6/1
Spectrometer equipped with an AquaX Aqueous Sample
Cell (30 L/cm volume). Spectra were obtained at a reson-
ant frequency 9.862 GHz, a microwave attenuation of 40
db, a modulation frequency 100 kHz, a conversion time
of 40.9 ms, a time constant of 1.280, and an average of 4
scans. Infrared spectra were obtained with a Nicolet FT-
IR spectrometer using KBr pellets containing approxima-
tely 5% (w/w) HA. For HA samples containing Mg2+, 8
g/L aqueous HA solutions with 1.0 × 10–2 M MgCl2 were
prepared and freeze dried. The dry material was used in
the KBr pellets. Standard 3-carbamoyl-2,2,5,5,-tetrame-
thyl-3-pyrrolin-1-yloxy free radical was used for spin
count calibration.
The acid content of HA samples was determined by
conductometric replacement titrations as described in
earlier reports [12-14]. Measurements were made with a
platinum conductivity probe manufactured by Vernier
(Beaverton, OR) and recorded with a Vernier LabQuest
module. HA solutions of 500 mg/L were dissolved in a
slight excess (ca. 5 µeq) of NaOH and titrated with 2 M
HCl.
Surface tension studies were performed on 500 mg/L
aqueous HA solutions, including various cations, using a
Fisher Surface Tensiomat Model no. 21, fitted with a
19-mm-diameter platinum-iridium ring. Solutions were
placed in a shallow glass dish of 50 mm diameter, and
the ring was inserted in the middle of the container to
avoid edge effects. The ring was raised through manual
operation of the torsion mechanism, and the tension
reading at the instant of surface detachment was noted.
All measurements were taken in triplicate at a tempera-
ture of 22˚C.
Dynamic light scattering experiments were performed
with a Coulter N4 Plus Submicron Particle Sizer equi-
pped with a 10-mW helium-neon laser (
 = 632.8 nm).
The concentration of aqueous humic acid (ca. 500 mg/L)
was adjusted to give scattering intensities in range 5 ×
104 - 1 × 106 counts per second. All measurements were
taken at a 62.6 detection angle and all reported sizes are
averages from 10 sequential runs of 300 s each.
3. Results and Discussion
Before The EPR spectra of HA obtained in this study
displayed a narrow line at an average value of g =
2.00361 (Figure 1), which is consistent with the charac-
teristic sharp resonance produced by the semiquinone
free radical [3,5,15].
Spectra obtained with HA metal complexes showed
similar peaks with slight variations in position and line
width, but notable differences in signal intensities (Fig-
ure 1, Table 1).
The data in Table 1 show that the addition of cations
generally increased the g-values of the resonances in the
respective EPR spectra. Jezierski and coworkers ascribed
the increased g-values in the presence of metal ions to
the formation of complexes with oxygen-rich groups in
HA [5]. Conductometric titration [12] revealed that the
humic material use in these studies had a carboxylic acid
content of ca. 5 meq/g, providing ample opportunity for
such interactions. They were confirmed by changes in
the carboxylate IR bands (not shown) upon the addition
of Mg2+: the 1710 cm–1 – C = O stretch and 1620 cm–1
asymmetric stretch coalesced into an unresolved band at
1629 cm–1, while the 1398 cm–1 HA band shifted to 1426
cm–1.

B. BAKAJOVÁ ET AL.
57
Figure 1. EPR spectra of 500 mg/L HA (top) and 500 mg/L HA + 1.0 × 10–3 M Mg2+ (bottom).
Table 1. EPR parameters for humic acids with addition of several cations.
Sample g value Line width (G)Free radical concentration
(spins/g × 10–17)
HA 500 ppm 2.00361 5.38 2.32
HA + 1 E-3 M Na 2.00418 3.68 3.00
HA + 1 E-3 M Li 2.00435 4.25 2.80
HA + 1 E-3 M Mg 2.00401 4.53 8.45
HA + 7 E-4 M Mg 2.00387 3.12 4.68
HA + 5 E-4 M Mg 2.00386 3.40 4.35
HA + 1 E-4 M Mg 2.00386 4.53 4.11
HA + 5 E-5 M Mg 2.00417 3.97 3.56
HA + 1 E-3 M Zn 2.00368 5.39 8.68
HA + 1 E-3 M Ba 2.00400 3.69 5.75
HA + 1 E-3 M Ca 2.00390 4.53 4.22
HA + 1 E-4 M Al 2.00415 4.53 3.13
HA + 1 E-4 M Sm 2.00447 3.97 2.64
HA + 1 E-4 M Ce 2.00433 3.69 3.01
HA + 1 E-3 M N2 2.00400 2.55 0.10
An interesting observation, also made by Golonka et al.
[11], was that the addition of 1.0 × 10–3 M Mg2+ to the
HA solution produced a more than 3-fold enhancement
of the EPR signal of the humates, indicating a stabiliza-
tion of the semiquinoid radical in the humic structure. In
view of the fact that Mg2+ and other divalent metal ions
are known to undergo bridging interactions with car-
boxyl groups on humates [10], this observation suggests
that such bridged structures were involved in the stabili-
zation of humic semiquinones.

B. BAKAJOVÁ ET AL.
58
Other divalent metal ions, Mg2+, Ba2+, Ca2+ and Zn2+,
were also found to give EPR resonance enhancements,
albeit to different degrees (see spin counts in Table 1). In
contrast, monovalent metal ions such as Na+ and Li+
produced little or no enhancement. The addition of a large
organic divalent cation, [CH33N-CH22-NCH33]2+, did
not enhance the humic EPR signal, but actually decrea-
sed it.
If the formation of bridged humates was the cause of
the observed EPR enhancements, then it should be ex-
pected that trivalent species such as Al3+, Sm3+, and Ce3+
would have similar effects. In fact, relatively small signal
increases (< 30%) were observed with these ions, but
their propensity to cause HA to precipitate at ionic con-
centrations greater than ca. 1.0 × 10–4 limited the ob-
servable effects to very dilute solutions. The data in Ta-
ble 1 show that low Mg2+ concentrations gave accord-
ingly small EPR signal enhancements, so it can be cau-
tiously inferred that the humic structures produced by
interaction with trivalent metal ions can also increase
humic EPR signals.
Bridging interactions between metal ions and (primar-
ily) carboxyl groups in humates can be both intra- and
inter-molecular. The latter case, especially, can lead to
precipitation at higher ionic strengths. It has been shown
[7] that this precipitation proceeds through a gradual
process of aggregation in which humic particles grow
from micellar to macroscopic size. The effect can also be
caused by 1:1 salts containing non-bridging (monovalent)
cations, which, when the ionic strength is high enough,
can shield the negative charges on humic polyanions
sufficiently to overcome mutual repulsion.
The question arises whether the apparent stabilization
of semiquinoid entities in humates in the presence of
metal ions depends entirely on bridging interactions, or
whether nonspecific aggregation effects also play a role.
Since the particle size of dissolved humates provides
one of the most direct measures of aggregation in HA
solutions, dynamic light scattering (DLS) measurements
were carried out on humic solutions with various additi-
ves. The results shown in Figure 2 pertain to solutions in
which no precipitation had taken place. It can be seen that
the addition of a monovalent metal ion did not produce
aggregate size changes [7], consistent with the view that
these ions do not bind humic polyanions together.
Figure 2 also shows that low concentrations of Mg2+
caused a small reduction in particle size, which has pre-
viously been ascribed to intramolecular contraction [8].
At a Mg2+ concentration around 5.0 × 10–4 M the average
particle size began to increase and continued to do so
until humic flocs appeared. In this progression of events,
EPR signal enhancement due to Mg2+ became noticeable
at a concentration of 4.0 × 10–4 M, i.e. slightly before the
HA particles began to grow significantly. This suggests
that radical stabilization was primarily predicated on
metal mediated intramolecular bridging.In the case of the
addition of Ce3+ the effect was greatly compressed (Fig-
ure 2) and proceeded to intermolecular aggregation at
low cation concentrations, quickly followed by precipita-
tion. As noted above, this left room for the observation
of only minor EPR signal enhancement.
Surface tension measurements, shown in Figure 3,
further illustrate the relation between the state of cation
induced humic aggregation and EPR signal enhancement.
The data for Mg2+ show a gradual decrease in surface
tension with cation concentration, which has previously
been ascribedto the formation of humic amphiphiles [11]
as the cations bind with and neutralize part of the anionic
(carboxyl) groups in the humate. This regimen coincides
with the conditions under which EPR enhancement was
observed. At higher Mg2+ concentrations, pseudomicellar
aggregation led to the removal of humate from the air-
water interface and a commensurate increase in surface
tension. EPR signal enhancement persisted in this region,
as it did with the larger humic particles shown in Figure
2. The surface tension data obtained with the addition of
Figure 2. Average particle size of HA in concentration 500 mg/L with addition of Li+, Mg2+ and Ce3+.

59 B. BAKAJOVÁ ET AL.
Figure 3. Variation of the surface tension of a 500 mg/L HA solution with addition of Mg2+ and Ce3+.
Table 2. EPR characteristic of HA in water and urea.
Sample g value Line width (G) Free radical concentration
(spins/g × 10–17)
HA 2.00361 3.35 2.32
HA + Mg2+† 2.00401 4.53 8.45
HA + urea* 2.00409 5.38 2.60
HA + urea + Mg2+ 2.00375 4.54 2.23
†1.0 × 10–3 M
*8 M
Ce3+ again showed the aggregation range to be truncated
by precipitation at low cation concentrations.
Urea is a well known chaotrope which, in high con-
centrations, disrupts the hydrogen-bonded network of
water, allowing macromolecules more structural freedom
and promoting protein extension and denaturation
[16,17]. It has been shown that the ability of urea to des-
tabilize hydrophobic aggregates extends to the break-up
of HA structures in aqueous solution [18]. The data in
Table 2 demonstrate that the disruption of these struc-
tures had a profound effect on the EPR signal. In solu-
tions containing only HA, the addition of urea made little
difference to the spin concentration, but the signal en-
hancement observed upon addition of Mg2+ disappeared
when urea was added. This adds further credence to the
contention that cation-bridged structures―which were
disrupted by urea―are essential to radical stabilization in
HA.
4. Conclusions
In conclusion it can be noted that diamagnetic species
such as alkaline earth metal ions and zinc ions exert
considerable influence on free radical concentrations in
humates by stabilizing semiquinone structures within the
humic framework. Disruption of the aggregates formed
by metal bridging interactions lead to a loss of radical
content and EPR enhancement.
5. Acknowledgements
B. Bakajová thanks the Ministry of Education, Youth,
and Sports of the Czech Republic for financial support
received to work at the University of Idaho.
6. References
[1] F. J. Stevenson, “Humus Chemistry. Genesis, Compo-
sition and Reactions,” 2nd Edition, Willey and Sons, New
York, 1982, pp. 195-220.
[2] N. Senesi and E. Loffredo, “The Chemistry of Soil
Organic Matter, Soil Physical Chemistry,” In: D. L.
Sparks, Ed., Soil Physical Chemistry, CRC Press, Boca
Raton, FL, 1999, pp. 239-370.
[3] M. Jerzykiewicz, A. Jiezierski, F. Czechowski and J.
Drozd, “Influence of Metal Ions Binding on Free Radical

B. BAKAJOVÁ ET AL.
60
Concentration in Humic Acids. A Quantitative Electron
Paramagnetic Resonance Study,” Organic Geochemistry,
Vol. 33, No. 3, 2002, pp. 265-268.
doi:10.1016/S0146-6380(01)00158-9
[4] M. Jerzykiewicz, J. Drozd and A. Jezierski, “Organic
Radicals and Paramagnetic Metal Complexes in Muni-
cipal Solid Waste Composts. An EPR Study and Che-
mical Study,” Chemosphere, Vol. 39, No. 2, 1999, pp.
253-268. doi:10.1016/S0045-6535(99)00107-1
[5] A. Jezierski, M. Czechovski, M. Jerzykiewicz and J.
Drozd, “EPR Investigations of Humic Acids Structure
from Compost, Soil, Peat and Soft Brown Coal upon
Oxidation and Metal Uptake,” Applied Magnetic Reso-
nance, Vol. 18, No. 1, 2000, pp. 127-136.
doi:10.1007/BF03162104
[6] A. Jezierski, M. Czechovski, M. Jerzykiewicz, I. Golonka,
J. Drozd, E. Bylinska, Y. Chen and M. Seaward, “Quan-
titative EPR Study on Free Radicals in the Natural
Polyphenols Interacting with Metal Ions and Other
Environmental Pollutants,” Spectrochimica Acta Part A,
Vol. 58, No. 6, 2002, pp. 1293-1300.
doi:10.1016/S1386-1425(01)00718-1
[7] N. Palmer and R. von Wandruszka, “Dynamic Light
Scattering Measurements of Particle Size Development in
Aqueous Humic Material,” Fresenius Journal of Ana-
lytical Chemistry, Vol. 371, No. 7, 2001, pp. 951-954.
doi:10.1007/s002160101037
[8] L. M. Yates and R. von Wandruszka, “Effects of pH and
Metals on the Surface Tension of Aqueous Humic Ma-
terials,” Soil Science Society of America Journal, Vol. 63,
No. 6, 1999, pp.1645-1649.
doi:10.2136/sssaj1999.6361645x
[9] R. R. Engebretson and R. von Wandruszka, “The Effect
of Molecular Size on Humic Acid Association,” Organic
Geochemistry, Vol. 26, No. 11-12, 1997, pp. 759-767.
doi:10.1016/S0146-6380(97)00057-0
[10] R. von Wandruszka, C. Ragle and R. Engebretson, “The
Role of Selected Cations in the Formation of Pseu-
domicelles in Aqueous Humic Acid,” Talanta, Vol. 44,
No. 5, 1997, pp. 805-809.
doi:10.1016/S0039-9140(96)02116-9
[11] I. Golonka, F. Czechowski and A. Jezierski, “EPR
Characteristic of Heat Treated Complexes of Metals with
Demineralised Humic Brown Coal in Air and Ammonia
Atmospheres,” Geoderma, Vol. 127, No. 3-4, 2005, pp.
237-252. doi:10.1016/j.geoderma.2004.12.005
[12] J. Riggle and R. von Wandruszka, “Conductometric
Characterization of Dissolved Humic Materials,” Talanta,
Vol. 57, No. 3, 2002, pp. 519-526.
doi:10.1016/S0039-9140(02)00052-8
[13] S. Arai and K. Kumada, “An Interpretation of the Con-
ductometric Titration Curve of Humic Acid,” Geoderma,
Vol. 19, No. 1, 1977, pp. 21-35.
doi:10.1016/0016-7061(77)90011-8
[14] S. Arai and K. Kumada, “Fractional Determination of
Functional Groups of Humic Acids by Conductometric
Titration,” Geoderma, Vol. 19, No. 4, 1977, pp. 307-317.
doi:10.1016/0016-7061(77)90072-6
[15] N. Senesi and M. Schnitzer, “Effect of pH, Reaction
Time, Chemical Reduction and Irradiation on ESR
Spectra of Fulvic Acid,” Soil Science, Vol. 123, 1977, pp.
224-234. doi:10.1097/00010694-197704000-00003
[16] H. Uedaira and H. Uedaira, “Role of Hydration of Poly-
hydroxy Compound in Biological Systems,” Cell Mole-
cular Biology, Vol. 7, 2001, pp. 823-829.
[17] R. R. Engebretson, T. Amos and R. von Wandruszka,
“Quantitative Approach to Humic Acid Associations,”
Environmental Science & Technology, Vol. 30, No. 3,
1996, pp. 990-997. doi:10.1021/es950478g
[18] J. Kislinger, F. Novak and J. Kucerik, “Role of Aromatic-
ity Degree in the Stability of Humic Substances,” In: I. V.
Perminova and N. A. Kulikova, Eds., Proceedings of the
14th Meeting of the International Humic Substances So-
ciety, Humus Sapiens, Moscow, Vol. 1, 2008, pp. 253-
256.