Soil Science and Plant Nutrition

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Relationship between viscosity and molecular

weight in an andosol humic acid

Masayuki Kawahigashi & Nobuhide Fujitake

To cite this article: Masayuki Kawahigashi & Nobuhide Fujitake (2001) Relationship between
viscosity and molecular weight in an andosol humic acid, Soil Science and Plant Nutrition, 47:2,
399-404, DOI: 10.1080/00380768.2001.10408402

To link to this article: https://doi.org/10.1080/00380768.2001.10408402

Published online: 04 Jan 2012.

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 Short Paper
Soil Sci. Plant Nutr., 47 (2), 399-404, 2001 399

Relationship between Viscosity and Molecular Weight

in an Andosol Humic Acid

Masayuki Kawahigashi 1 and Nobuhide Fujitake*

The Graduate School of Science and Technology and • Faculty of Agriculture,

Kobe University, Kobe, 657-8501 Japan

Received June 6, 2000; accepted in revised form March 27, 2001

Viscosity and molecular weight of particle size fractions obtained from an

Andosol humic acid (HA) were determined. Viscosity was determined both in
solutions with and without the addition of 0.1 M N aCI (Cs 0.1 and Cs 0, respec-
tively) at pH 7.0. Polyelectrolytic character was observed in the particle size
fractions based on the changes in the concentration dependence of reduced
viscosity with NaCI concentrations. The use of a Cs 0.1 solution was suitable for
the determination of the values of intrinsic viscosity ([ 7) J) of the particle size
fractions. The [7)J value increased with increasing weight average molecular
weight (Mw ) determined by gel permeation chromatography (GPC), and ranged
from 4.3 to 12.9 X 10- 3 (L g-l) in the Cs 0 solution and from 3.5 to 6.6 X 10- 3 (L
g-l) in the Cs 0.1 solution. A linear relationship between [7) J in the Cs 0.1
solution and Mw on a logarithmic scale, which was similar to the Mark-
Houwink equation, was observed. The value of the constant a calculated from
the relationship, which reflected the polymer morphology, was 0.75 in the Cs 0.1
solution, and it corresponded to a polymer with flexible chains.
Key Words: GPC, homologous series, humic acid, polyelectrolyte, viscosity.

Humic substances (HS) extracted from soils show a wide distribution of molecular
weight or molecular size. Studies on the physico-chemical properties of particle size fractions
of HS have provided significant information about the relationship between particle size and
structural properties. With increasing molecular weight or particle size, the contents of
aromatic structural units and carboxylic functional groups decreased, while those of saturat-
ed aliphatic structural units and phenolic functional groups increased (Tomikawa and Oba
1991; Swift et al. 1992; Kawahigashi et al. 1996; Fujitake and Kawahigashi 1998). Surface-
active properties also changed with increasing particle size (Hayano et al. 1982; Hayase and
Tsubota 1983; Kawahigashi and Fujitake 1998). The elucidation of the physico-chemical
properties of particle size fractions of HS is necessary to characterize HS. Although HS show
values of molecular weights ranging from thousands to tens of thousands (Hayes et al. 1989),
there have been few studies on the polymer properties of HS.
Generally, a series of polymers show a significant relationship between intrinsic viscos-
ity ([17J) and weight average molecular weight (Mw ) (Lovell 1989). A constant calculated
from both parameters characterizes a series of polymers. Few studies have been carried out

1 Present address: College of Bioresource Science, Nihon University, Fujisawa, 252-8510 Japan.
400 M. KA WAHIGASHI and N. FUJITAKE

to analyze the relationship between both parameters in HS. In the current study, emphasis
was placed on the analysis of the relationship between [17] and Mw determined by gel
permeation chromatography (GPC), using particle size fractions separated from a humic
acid (HA), along with examining the polymer characteristics of a HA.

Theory
The relationship between the viscosity average molecular weight and [17] in a series of
polymer samples was reviewed by Lovell (1989). The following empirical equation designat-
ed as Mark-Houwink equation was proposed by many workers,

where My is the viscosity average molecular weight, and K and a are constants for a given
polymer/solvent/temperature system. The a value is related to a polymer morphology: 0.5
to 0.8 for flexible chains such as polystyrenes, polyesters, and polyamides, which consist of
long chains of small identical subunits; 0.8 to 1.0 for stiff molecules such as cellulose
derivatives and DNA; and 1.0 to 1.7 for highly extended chains (e.g. polyelectrolytes in low
ionic strength solutions). K values for flexible chains are generally in the range of 10- 6 to
10- 3 L g-l and tend to change with a. My can only be obtained from [17] measured in the
system where the K and a values are known. On the other hand, the K and a values are
determined from [17] for a series of polymer samples with a known number average
molecular weight (Mn ) or Mw , and Mw is preferable to Mn- For a series of polymers, the plot
of log [17] against log Mw can be fitted to a straight line, from which the K and a values
are determined.

Materials and methods

Humic acid. The humic acid was extracted from Sugadaira soil (Umbric Andosol) in
Nagano Prefecture, Japan and was obtained in a powder form by freeze-drying after
elimination of contaminants such as non-humic substances, clays, and ions in the same way
as described in our previous paper (Kawahigashi and Fujitake 1998). Four particle size
fractions separated by the sequential GPC with the preparative gel columns utilized in our
previous study (Kawahigashi and Fujitake 1998) were used for this study. The molecular
weights of these fractions were roughly estimated to be 3, 10, 30, and 100 kilodalton (kD)
based on the exclusion limit of each preparative GPC column determined by using dextran
as a standard. Each fraction was designated as 3K, 10K, 30K, and lOOK, respectively.
Molecular weight determination by GPC. A 80 mg aliquot of HA was suspended
in 2.5 mL of 0.05 M NaOH and 200,uL of 1 M NaOH was added to the suspension. Then the
suspension was sonicated to dissolve HA completely. The solution pH was adjusted to 8.0
with dil. NaOH. The volume of the solution was adjusted to 5 mL with distilled water firstly
and to 10 mL with a 0.04 M Na 2 C0 3 -O.04 M HCl buffer solution (pH 8.0) containing 40%
methanol. The solution was diluted to reach a concentration of 0.8 g L-l with a 0.02 M
Na 2 C0 3 -O.02 M HCl buffer solution (pH 8.0) containing 20% methanol. A 1 mL aliquot of
this diluted solution was filtered through a 0.2,um membrane filter and a lO,uL aliquot of
the filtered solution was applied to the GPC system, which consisted of a plunger pump, an
ultraviolet (detection at 280 nm) detector, and a refractive index detector, equipped with two
columns (both being 0.75 cm in diameter and 30 cm in length) of Shodex 803HQ (exclusion
limit of 1.0 X 10 5 ) and 805HQ (exclusion limit of 4.0 X 10 6 ) in series, preceded by a guard
column. Pullulan standards with different molecular weights were used for the establishment
 Relationship between Viscosity and Molecular Weight in Humic Acid 401

of a linear calibration tentatively. The flow rate was set at 0.6 mL min-I.
Viscosity measurement. A 100 mg aliquot of each HA fraction was dissolved in a
small amount of 1 M NaOH and diluted to 5 mL, equivalent to 20 g L -1, after the pH was
adjusted to 7.0. The HA solution was further diluted to 10 mL with a buffer solution (0.2
M Na4P207-0.2 M H 4P 20 7, pH 7.0) for the solution without salts (Cs 0) and with the same
buffer solution containing 0.2 M N aCl to prepare the 0.1 M N aCl (Cs 0.1) solution finally.
These solutions were filtered through a 0.2 Jim membrane filter. A quantity of 8 mL of the
filtered solution was applied to an Ubbelohde-type viscometer with a flow time of 260 s for
distilled water at 25 ±0.01"C. Concentrations of the solution were changed successively by
the addition of a suitable volume of buffer solution (0.1 M N a 4P 20 7-O.1 M H 4P 20 7, pH 7.0)
or the buffer solution containing 0.1 M NaCl, and the viscosity measurement was performed
over a wide range of concentrations from 1 to 10 g L -1. The measurement of the flow time
which passed through two etched marks from upper to lower for each concentration was
repeated 5 to 10 times and data within 0.3 s differences in flow time were averaged. Reduced
viscosity, r;sp/ e was represented by Huggins equation as follows:
r;sp/ e = [r;] + k [r; Fe
where, [r;] is the intrinsic viscosity (L g-I), e is the sample concentration, and k is the
Huggins constant (Lovell 1989). The [r;] value was estimated by extrapolation to a zero
concentration of the linear part of the concentration dependence of r;sp/ e. Specific viscosity,
r;sp was calculated by the following equation.
r;sp = (ts - to)/ to
where ts and to are the flow time of the HA solution and buffer solution, respectively.

Results and discussion

The relationships between the r;sp/ e values and the concentrations of particle size

~.02 0.02
3K • 30K
::;--
•
be
::::,
u 0.01
.........•..•...•....•.•
•
it ...•..•.. !I! .
i=" 8l1i'l
••• I' .•.•.•:.:......
:.•:.•·:iI·: .•.•.•.• 'lII

o 5 10 15 o 5 10 15
Concenlration (g L-J) Concentration (g L-J)

0.02 • 0.02
10K
• lOOK
::;--
be • ~be ••
::::, ::::,
•
" •••• IIP •••• ••••••
...•....• • ····e····.~._ .. ~ ....• Fig. 1. Relationship
~
~ 0.01
i="
.... ~ .
i="
0.01

o 5
Concentration (g L-l)
·············ii············..
between reduced viscos-
ity (T/sp/ c) values and
concentrations of parti-
cle size fractions. e, Cs
10 15 o 5 10 15 o solution; ., Cs 0.1
Concentration (g L- 1) solution.
402 M. KAWAHIGASHI and N. FUJITAKE

fractions in the Cs 0 and Cs 0.1 solutions are shown in Fig. 1. In the Cs 0 solution, except
for 3K, the rJsp/ c values tended to increase with decreasing concentrations under ca. 5 g L-I.
This increase in the rJsp/ c values in the low concentration range may be attributed to the
expansion of HA molecules caused by repulsion between ionized functional groups (Budd
1989).
In the case of HAs, dissociated carboxylic functional groups contribute significantly to
intramolecular repulsion. Therefore, the viscosity measurement in the Cs 0 solution was not
suitable for determining [rJ]' On the contrary, in the 3K fraction, the values of rJsp/ c in the
Cs 0 solution decreased with decreasing concentrations. The differences in the viscosity
between 3K and the other fractions could be attributed to differences in the structural
properties. The content of a saturated aliphatic hydrocarbon influencing the changes in the
shape of HA molecules was lower in the 3K fraction than in the other fractions based on
the 13C NMR spectra (Fujitake and Kawahigashi 1998).
The values of rJsp/ c in the Cs 0.1 solution were lower than those in the Cs 0 solution
for every concentration. Changes in the rJsp/ c values with the concentrations were less
appreciable in the Cs 0.1 solution than in the Cs 0 solution. The concentration dependence
of rJsp/ c in the Cs 0.1 solution is likely to be brought about by the reduction of repulsion
between ionizable functional groups by a counter ion, Na+. It appeared that the HA
molecules behaved like nonionic polymer solutes without solvent influence. The viscosity
measurement in the Cs 0.1 solution was considered to be suitable for the characterization of
HA polymer properties. It was reported that the viscosity measurement of the HA solution
without intramolecular repulsion by functional groups could be achieved in aN aCI solution
(Ghosh and Schnitzer 1980; Visser 1985) and in 0.1 M NaOH (Kumada and Kawamura
1965).
The [rJ] values (L g-I) of the fractions ranged from 4.3 to l2.9X 10- 3 in the Cs 0
solution and from 3.5 to 6.6 X 10- 3 in Cs 0.1 solution (Table 1). These values were similar
to those obtained by Visser (1985). The decrease in the [rJ] values by coexisting salts was
considerably less pronounced in the 3K fraction than in the other fractions. The [rJ] values
in both solutions increased with increasing particle sizes. Mw also increased with increasing
particle sizes, and ranged from 26 to 59 kD (Table 1).
The relationships between log Mw and log [rJ] are shown in Fig. 2. In the Cs 0 solution,
no linear relationship was clearly observed. On the other hand, a linear relationship was
observed in the Cs 0.1 solution. The relationship between the two parameters was similar to
the Mark-Howink equation which is well recognized in various types of polymers.
Homologous series of polymers in the HA might be brought about by the differences in the
proportion of aliphatic structures, which increased with increasing particle size of HA
(Kawahigashi et al. 1996; Fujitake and Kawahigashi 1998).
The slope of equations in Fig. 2, a, reflecting the polymer morphology, was 1.37 and

Table 1. Intrinsic viscosity ([17] X 10') and weight average molecular weight (Mw ) of each fraction.

Fractions M w (kD)
Cs 0 Cs 0.1
3K 4.32 3.45 26.3
10K 11.55 4.42 42.6
30K 11.57 5.61 56.8
lOOK 12.86 6.55 59.0
Cs 0, HA solution without NaC!; Cs 0.1, HA solution with 0.1 M NaC!.
 Relationship between Viscosity and Molecular Weight in Humic Acid 403
-1.80 y=1.37x-B.39
R2 =O.892

Fig. 2. Relationship between logarithmic weight aver-

age molecular weight (log M,v) and logarithmic intrinsic
-2.60L...-----------
4.40 4.60 4.80 viscosity (log [71 J). • , Cs 0 solution; _, Cs 0.1 solution.
logMw • Significant at 5% level.

0.75 in the Cs 0 and Cs 0.1 solutions, respectively. Morphology of HA estimated from these
values consisted of highly extended chains in the Cs 0 solution, and flexible chains in the Cs
0.1 solution. These a values were different from those utilized in past studies for calculating
Mv of HAs and FAs, 0.65 and 0.67 (Chen and Schnitzer 1976; Ghosh and Schnitzer 1980,
1981; Rey et al. 1996; Gonet and Wegner 1997).
Our study suggested that the analysis of the relationship between the molecular weight
and viscosity is an effective method for obtaining the a value of HAs. The chemical and
skeletal structure of polymer molecules affects their hydrodynamic behavior (Lovell 1989).
Length of aliphatic chains, number of branches in the aliphatic structure and content of
carboxylic functional groups largely influence the viscosity of the HA molecules. The a
value is a major physical parameter of HAs reflecting their structural properties. Therefore,
further studies on a values of HAs from various origins should be carried out.

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