Colloid Journal, Vol. 64, No. 6, 2002, pp. 681685. Translated from Kolloidnyi Zhurnal, Vol. 64, No.

6, 2002, pp. 756761.

Original Russian Text Copyright 2002 by Bratskaya, Avramenko, Sukhoverkhov, Schwarz.

Flocculation of Humic Substances

and Their Derivatives with Chitosan
S. Yu. Bratskaya*, V. A. Avramenko*, S. V. Sukhoverkhov**, and S. Schwarz***
* Institute of Chemistry, Far East Division, Russian Academy of Sciences,
pr. Stoletiya Vladivostoka 159, Vladivostok, 690022 Russia
** Pacific Fisheries Research Center, tupik Shevchenko 4, Vladivostok, 690950 Russia
*** Institute of Polymer Research, Dresdene. V. Hohe Str. 6, D-01069 Dresden, Germany
Received June 29, 2001

AbstractThe efficiency of the flocculation of humic compounds and their derivatives with chitosan was studied as a function of pH, concentrations of chitosan and dissolved humic compounds, and also the presence of
inorganic coagulants. Samples of humic compounds were studied by the methods of potentiometric titration
and gel permeation chromatography. The molecular weight distributions and the spectra of ionization constants
of their functional groups were calculated. The flocculation efficiency of chitosan with respect to humic compounds is much higher than that of polyacrylamide.

INTRODUCTION
Natural polymers chitin and chitosan produced from
the shells of sea Crustacea and mycelium of some
mushrooms for the last decades have been the objects of
persistent attention. Because of the presence of an amino
group and a large number of hydroxy groups in each
monosaccharide unit, chitosan (deacetylated chitin) has a
high adsorption ability with respect to some metal
ions [1, 2], dyes [3], polyphenols [4], and other organic
compounds that explains its wide use in pharmacology,
biochemistry, biotechnology, medicine, and agriculture. Waste and drinking water treatment are extremely
promising fields of chitosan use. In addition to good
complexing properties with respect to metal ions, chitosan has a high coagulation efficiency and can be used
for bonding and subsequent precipitation of humic
compounds and surfactants polluting waters of various
origins.
At present, the following methods are predominantly used to purify natural waters from humic compounds: adsorption on kaolin [5] or activated carbon
[6], ozonization [7], and coagulation with iron and aluminum salts [8], as well as with synthetic polymers [9].
The use of inorganic polyelectrolyte coagulants results
in adequate purification of waste and natural waters
from organic substances; i.e., the coagulation followed
by filtration shows the efficiency of 7590%, as judged
by the dry residue. At the same time, the efficiency of
this method estimated by color index and turbidity is
substantially lower, 4070%. Although many synthetic
polymeric flocculants are more efficient than aluminum
and iron salts, they also do not provide a high degree of
purification of some natural waters. Furthermore, these
flocculants can pollute the environment, because of
their nonbiodegradability.

Chitosan and its derivatives are well known as flocculants of kaolin and clay suspensions, and chitosan mixture with bentonite is used to purify wastewater [10, 11].
Undissolved chitin and chitosan were shown [12] to be
good adsorbents with respect to humic compounds and
can extract from water up to 28.8 mg of these compounds per gram of the sorbent. However, being used as
a flocculant, chitosan is much more efficient and can
decrease in the color index of purified natural waters
almost by 100%.
In this work, the flocculation of humic compounds
and their derivatives with chitosan was studied, and the
effect of pH and the concentrations of the flocculant
and dissolved humic compounds on the efficiency of
water purification was analyzed.
EXPERIMENTAL
Characteristics of the Objects for the Study
We used chitosan with the degree of deacetylation
of approximately 75% and average molecular weight
100 kDa (a single fraction, as determined by chromatography) provided by the Pacific Institute of Bioorganic Chemistry, Far East Division, Russian Academy
of Sciences. A stock solution was prepared by dissolving chitosan (2.5 g) in 0.1 M HCl (250 ml) upon heating
in a water bath at 80C.
Humic compounds were extracted from peat (provided by V. M. Dudarchik, Institute of Ecology, Minsk,
Belarus) with concentrated ammonia at 1 : 20 solid :
liquid ratio. In order to remove the excess ammonia, the
filtration of the obtained solution was followed by its
partial evaporation. The concentration of humic compounds in the solution was determined by the dry residue. Sodium salts of humic acids were prepared by pre-

1061-933X/02/6406-0681$27.00 2002 MAIK Nauka /Interperiodica

682

BRATSKAYA et al.

cipitating humic compounds with concentrated hydrochloric acid followed by the precipitate centrifugation
and dissolution in a sodium hydroxide solution. The
end sample consisted of three fractions with average
molecular weights >250, 189, and 45 kDa, as determined by chromatography.
Amides of humic compounds were prepared by
heating humate solution in concentrated ammonia in a
sealed steel autoclave for 4 h at 140C. Concentration
of the obtained humate solutions varied from 0.2 to
0.8%. Solutions of required concentrations were prepared by diluting the initial solutions with distilled
water.
We also used sodium humate H1 675-2, Lot 31620-99
(Aldrich). This sample consisted of three fractions with
average molecular weights > 250, 126, and 18 kDa
determined by chromatography.
Chromatographic Study of Chitosan
and Humic Compounds
A Shimadzu LC-6A liquid chromatograph (Japan)
equipped with an LC-6A pump, a DGU-2A helium
stripping vessel, an SIL-6B automatic injector, an
STO-6A thermostat, an SPD-6AV ultraviolet detector,
a RID-6A refractometric detector, and a SCL-6B controller, was used in this work. Chromatograms were recorded
and processed with a CHROMATOPAC C-R4A integrator (Shimadzu, Japan). The separation was performed
in a Shodex Asahipak GS-520H column (250 7.6 mm;
sorbent particle size, 9 m) thermostated at 45C.
The chromatographic system was calibrated using
1 mg/ml solutions of standard samples of T-series dextrans (Pharmacia Biotech) with molecular weights of
10, 20, 40, 70, 80, 110, 250, 500 and 2000 kDa. Molecular weight and molecular weight distribution were calculated using the 7GPC software package.
Molecular weights were determined using 1 mg/ml
solutions prepared in the eluent and prefiltered through
a Kurabo 25Al membrane filter with the pore size of
0.45 m. Humic acids were dissolved in distilled water,
and chitosan, in 0.2 M NaNO3 solution containing 1%
of acetic acid (pH 2.9). The elution rate was 1 ml/min.
Chitosan was detected with the differential refractometer, and humic substances, by UV absorption at a wavelength of 263 nm.

burette was operated by means of an apparatus interface.

Sodium hydroxide solutions were used as titrants.
Hydrochloric acid was preliminarily added as a background electrotype to the samples containing up to 2 g/l
of humates in order to bring their pH to 2. The titration
was performed in a pH range from 2 to 10.5. Processing
of the titration curves by the method of density functions [13] made it possible to present the functional
groups of humic compounds as continuous spectra of
ionization constants.
The Study of Flocculation
In order to study the degree of color removal as a function of chitosan concentration, from 0.1 to 2.0 ml of 1%
chitosan solution in 0.1 N HCl or 0.1% chitosan solution in 0.01 N HCl was added into conical flasks containing humate solutions (40 ml) with concentrations
from 100 to 500 mg/l. Then, the solution pH was
adjusted to 7 by adding several drops of a NaOH or HCl
solution. To provide complete flocculation, the solutions were allowed to stand for 24 h. Then, the solutions
were filtered trough blue ribbon paper filters and the
transparency was determined with a UV-VIS 1201 spectrophotometer (Shimadzu) at a wavelength of 470 nm.
The degree of color removal Q was calculated for each
solution by the formula
Q = 100(D0 D)/D0,
where D0 and D are the optical densities of the initial
humate solution and the filtrate, respectively. The
degree of color removal was also studied as a function
of pH at different humate-to-chitosan ratios and of chitosan concentration in the presence of iron(III) ions.
Determination of the Effective Charge
of Colloidal Particles
Colloidal titration was performed with a system
consisting of a Mtek PCD 02 charge detector and a
Mettler DL21 automatic titrator. The system was operated by a personal computer. Solutions of an anionite
PDADMAC (polydiallyldimethylammonium chloride)
and a cationite (sodium polyethylenesulfate) with concentrations of 103 mol/l were used as titrants. Solutions
of humates and chitosan were kept for 15 min prior to the
titration. Titrated sample volumes were equal to 10 ml.

Potentiometric Titration of Humic Compounds

Humic compounds were titrated with an automatic
titration system consisting of a Radelkis OP-211/1
pH-meter with a measurement accuracy of 0.01 pH
units, a Radelkis OP-0808F combined glass electrode,
a sealed Teflon measuring cell, a magnetic stirrer, a
thermostat, a Radelkis OP-930/1 automatic burette, and
an IBM Pentium-100 personal computer. Indications of
the pH-meter and the burette were registered and the

RESULTS AND DISCUSSION

The study of chitosan flocculating properties in
solutions of humic compounds is of interest for the
removal of various metals from industrial wastewater.
Using humic compounds as a sorption reagent and chitosan as a flocculant, we can concentrate valuable metals from large volumes of wastewater and purify the latter from humic compounds.
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FLOCCULATION OF HUMIC SUBSTANCES AND THEIR DERIVATIVES WITH CHITOSAN

Q, %
100
1
2
3
4
5

80
60
40
20
0

10

12
pH

Fig. 1. Plots of the degree of color removal from humic

compound solutions versus pH at different chitosan-to-humate
ratios: (1) 0.2, (2) 0.5, (3) 1, (4) 2, and (5) 4.

In the presence of chitosan, humic acids can be practically completely (95100%) removed from solution
at pH 7 (Fig. 1). Moreover, the efficient flocculation is
observed in a pH interval from 6.5 to 8.0 at chitosan-tohumate ratios ranging from 1 : 1 to 1 : 3. As is seen, an
excess of chitosan stabilizes the colloid of humic compounds, thus causing a decrease in the efficiency of
water purification. In the case of chitosan deficiency,
the most substantial decrease in the solution transparency is observed at lower pH values that is most likely
associated with the precipitation of protonated humic
compounds rather than humatechitosan complexes.
Nevertheless, in the absence of chitosan, the precipita-

683

tion of humic compounds with an efficiency of at least

70% is possible, but at low pH values. For example, the
studied humates extracted from peat precipitated only
at pH 0. Obviously, when it is required to precipitate
humates used as sorbents of metal ions, extremely low
pH values are unacceptable because of technological
reasons. Furthermore, in highly acidic media, humic
compounds are protonated and lose their complexing
properties, thus making their use inefficient.
The amount of chitosan necessary for the efficient
flocculation of humic compounds is governed first of
all by the charge density of humates: the higher the negative particle charge the larger amount of positively
charged chitosan is required for its compensation. As is
seen from Fig. 2, humates require larger amounts of
chitosan for their complete precipitation from solutions
than their amides.
Potentiometric titration showed that the initial
humic acids contain sufficiently large number of carboxyl groups with pKa values ranging from 3.5 to 5.5,
whereas these groups are absolutely absent in amides
(Fig. 3). This change in the composition of the functional groups seemingly decreases the negative charge
density of humic compounds and, consequently, lowers
their colloidal stability with respect to positively
charged chitosan. The sodium humate sample (Aldrich)
also contains a small number of functional groups with
pK lower than 5 (Fig. 4), and for its efficient flocculation a somewhat smaller amount of chitosan is required
as compared to the precipitation of the initial humates
extracted from peat (Fig. 5).
Note that an unfiltered solution of the Aldrich
humate had markedly lower colloidal stability. The
addition of small amounts of chitosan caused the precipitation. Then, however, the flocculation efficiency

0.020

Q, %
100
80

0.015

60
1
2
3
4

40
20

10 20 30 40 50 60 80 90
C 103, %

Fig. 2. Plots of the degree of color removal from solutions

of (1, 2) humic compounds and (3, 4) their amides versus
chitosan concentration. Initial solution concentration: (1) 100,
(2) 500, (3) 100, and (4) 400 mg/l.
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0.010
2
0.005

10

12

14
pKa

Fig. 3. The spectra of ionization constants of (1) humic

acids and (2) their amides.

684

BRATSKAYA et al.

0.020

Q, %
100
80

0.015

1
2
3

60
0.010

40
20

0.005

0
4

10

12

14
pK

Fig. 4. The spectrum of ionization constants of the humate

(Aldrich).

20

40

60

80
C 103, %

Fig. 5. Plots of the degree of color removal from the humate

(Aldrich) solutions versus chitosan concentration. Concentrations of the humate solutions: (1, 2) 100 and (3) 500 mg/l.
Solutions 2 and 3 were prefiltered.

Charge density, mmol/l

0.4

Q, %
100

1
2
3
4
5
6

0.3

80

0.2
60
0.1
1
2
3

40
20

0
0.1
0.2

5
6
7
C 103, %

10

12
pH

Fig. 6. Dependences of the degree of color removal from the

solutions of (1, 2) humic compounds and (3) their amides on
chitosan concentration in the presence of (1, 3) 50 and
(2) 100 mg/l of iron(III) salts. Humic compound concentrations: (1, 3) 100 and (2) 400 mg/l.

Fig. 7. Dependences of effective charge density, as determined by colloidal titration, on pH for the humatechitosan
systems. Humic compound concentrations: (2) 0 and (1, 36)
50 mg/l. Chitosan concentrations: (1) 0; (2, 4) 50; (3) 20;
(5) 30; and (6) 10 mg/l.

drastically decreased, and a further increase in the flocculant amount led to the stabilization of the colloidal
system. This phenomenon was not observed for humate
solutions prefiltered through the blue ribbon paper
filters (Fig. 5).

to the formation of a denser precipitate at lower chitosan concentrations in the cases of both humic acids and
their amides. Note that, in the case of the studied humic
compounds with a high content of carboxyl groups, the
efficiency of the precipitation only with iron salts is not
higher than 50% even at large amounts of coagulant.

Trivalent iron salts substantially influence the efficiency of humic compound flocculation with chitosan.
It is known that precipitates resulted from the humic
compound coagulation with inorganic electrolytes
including iron salts often have a loose structure. They
are characterized by high filtration resistance, especially at high concentrations of humic compounds in
waters to be purified. At the same time, the use of chitosan in combination with trivalent iron compounds leads
to an increase in the flocculation efficiency (Fig. 6) and

In addition to the charge of colloidal particles,

molecular weights of both flocculant and precipitated
humic compounds substantially influence the flocculation efficiency. Chitosan used in this work belongs to
flocculants with low or medium molecular weights and
sufficiently high charge density. Flocculation by the
mechanism of the charge compensation due to the
adsorption of an oppositely charged polymer on dispersed particles is more typical of such polyelectroCOLLOID JOURNAL

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FLOCCULATION OF HUMIC SUBSTANCES AND THEIR DERIVATIVES WITH CHITOSAN

Q, %
100
80
60

1
2
3

40
20
0

20

40

60
80
C 103, %

Fig. 8. Plots of the degree of color removal from solutions of

humic compounds (100 mg/l) versus flocculant concentration: (1) chitosan, (2) PAA, and (3) PAA + 50 mg/l of Fe(III).

685

addition to a high-molecular-weight fraction, both samples contain quite large amounts of low- and mediummolecular-weight polymers, thus making their colloidal solutions highly stable at the high charge density on
macromolecules due to the dissociation of carboxyl
groups.
We can assume that the use of PAA makes it possible to remove a certain fraction of high-molecularweight humic compounds from a solution, while the
flocculation with synthetic polyelectrolyte by the bridging mechanism appears to be inefficient for the removal
of the low-molecular-weight fraction.
ACKNOWLEDGMENTS
This work was supported by INTAS (grant
YSF 2000-38) and the Ministry of Science and Art of
Saxon, Germany (grant no. 4-7531.050-04-821-01/11).
REFERENCES

lytes. This mechanism implies the efficient flocculation

in a quite narrow concentration range [14], outside
which particles acquire an opposite charge preventing
the flocculation.
Figures 1 and 2 demonstrate that chitosan is an efficient flocculant within a wide concentration range,
although its excess somewhat decreases the flocculation efficiency. As is seen from Fig. 7, the effective negative charge of humates considerably decreases already
at 1 : 5 humate-to-chitosan mass ratio. The efficiency of
a decrease in the charge of humic compounds is probably associated with a much higher positive charge density
of chitosan molecules that is determined by the regular
structure of this polyelectrolyte (see curves 1 and 2).
Comparative studies of polyacrylamide (PAA) and
chitosan efficiency in the flocculation of humic compounds showed that PAA provides no more than 10%
decrease in the solution transparency under the conditions corresponding to almost 100% chitosan efficiency
(Fig. 8). Combination of PAA and trivalent iron salts
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electrolytes. Moreover, an increase in this polymer concentration stabilizes the humic compoundsFe(III)
colloids and decreases the flocculation efficiency.
As was mentioned above, according to chromatographic analysis, humate extracted from peat and the
sodium humate (Aldrich) are characterized by a complex (three-fraction) molecular weight distribution. In

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2002

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