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Surface active properties of soil humic acids
The Science Elsevier
SURFACE
K.
of the Total Environment,
Science
Publishers
ACTIVE
B.V.,
PROPERTIES
YONEBAYASHI
and
T.
62 (1987) 55-64
Amsterdam
OF
- Printed
SOIL
HUMIC
55
in The Netherlands
ACIDS
HATTORI
Faculty of Agriculture, 606 kyoto (Japan)
Kyoto
Prefectural
University,
Shimogamo,
Sakyo-ku,
SUMMARY For gel permeation chromatography of humic acids, use of a neutral phosIt was determined that the phate buffer containing 2 M urea is recommended. excluded fraction increased with an increase in standing time of sample in buffer solution before injection into the column. From the gel permeation chromatography of mixed samples of excluded and retarded fractions and meait was concluded that the surement of the surface tension of each fraction, reason is that large humic molecules tend to associate with small ones to form micelle-like aggregates. Various types of humic acids were subjected to the measurement of surface tension and gel permeation chromatography. Humic acid containing a large amount of excluded fraction showed high surface activity. The relationships between surface activity and structural properties of humic acid are discussed.
INTRODUCTION Gel
permeation
humic
chromatography
materials
between
(refs.
humic
static
substances
and
interactions We have
and
alkaline
In
this
surface
MATERIALS Soil
an of
samples
and
humic soil
to
applied
have
to
overcome
such
as
the
studies
of
interactions
adsorption
and
electro-
gel
can
6).
In
acids
be
to
Sephadex
gel
by
use
eliminated
practice.
the
however,
of
alkaline
materials. buffer
acid
of
humic
behavior (ref.
appropriate humic
of
in
humic
solution
this
was
buffer
acid
(refs.
examined
solution
was
as
eluent.
related
to
the
7.8).
METHODS
Thirty-five selected
extensively
problems
for
properties
AND
materials,
eluent
undesirable
active
gel
adsorption as
research,
researchers
4,5).
that
association
been
and
adsorption
solution
are
the
the
found
urea
conditions
and
(refs.
studied
materials
has
l-3),
include
types
of
soil
and
Table
1.
Of
these
Humic
acids
IHSS
procedure.
were
0048-9697/87/$03.50
acids
samples
were
used
samples
that
differed
land
use.
samples, extracted
0 1987
A brief only from
Elsevier
as
dried
in
SA-1
these
soils
soils.
degree
description
two,
Science
air
of and and
Publishers
KP-1. purified
B.V.
These
of
humification
the
samples
were
used according
soils
were and
is in
given
in
Experiment to
the
1.
56
Table
1.
Soil
Classification
of
classification
soil
Soil
Andosol
samples
No.
(land
soil
SA-2(u,
JP),
SA-3(p,
JP),
TG-3(u,
JP),
MZ-1-l(v,
JP),
or
Muck
or
Brown
Jahgaru Peat
forest
and
THAI),
GF-4(b,
JP),
SG-3(p,
JP),
soil
NM-E(f,
JP),
KPF-l(f,JP),
U:
For
the
gel
JP),
was
M phosphate
between
4.7
into
borate
11.2
JP) JP),
B-l(f,
SG-2(p,
JP),
KP-l(p,
SA-4(p,
JP),
MGV-3(v,
v: virgin, Brazil.
b:
KPF-3(f,
JP),
B-lO(v,
BZ)
BZ), JP),
KP-2(p,JP),
JP)
buried.
the
pH
were
in
the
column.
An
effluent
tension
solution
(pH
of 7)
column
(2
containing
effects
prepared
surface
Sephadex
a glass
buffers
when
were
into
buffer
paddy, BZ:
THAI),
chromatography,
packed or
-
(0.2%)
injected The
upland, p: Thailand,
SG-4(b, KPF-E(f,
TJ-4(f.
JP), JP),
SG-l(p,
permeation
and
tions
JP)
procedure
material, 0.1
MZ-1-9(b,
THAI)
soil
THAI:
Measurement
T-209(p, JP)
soil
TG-l(p.JP),
JP).
Mongol)
ON-2(p,
Gley
forest, Japan,
MOGL(v,
JP),
NG-5(p, f: JP:
JP),
ON-l(v,
KPF-4(f, Grey
MZ-1-6(b,
JP).
Argentina) USA),
PS-16(u,
Mahji
location)
JP),
WU,
Grumusol
and
SA-l(f,
sus-l(u,
Chestnut
use
TG-P(v, MZ-1-3(b, Chernozem
used.
humic
acid
containing
was
cm).
2 M urea, being
same
G-75 X 45
whose
solution
as (400
solutions
the
eluent
nm)
was in
measured
by
gel
solu-
immediately as
0.1
varied
acid
and used
the
were
were
Humic
dissolved was
as eluents
pHs
investigated.
monitor
2 M urea
used The
detector.
M phosphate Wilhelmy
method. Data
processing Cluster
analysis
the
similarity
as
a function
the
SAS
Kyoto
was in
of
RESULTS
Analysis (ref.
AND
Experiment
adopted
pattern
to of
concentration.
(Statistical
University
For
the
classify decrease We used
System)
the in
at
soils
used
surface
the
computer
the
Data
on
tension
the of
program Processing
basis humic
of acid
contained Center
in of
9).
DISCUSSIONS 1
gel
and
gel
and
borate
must
permeation be buffers
chromatography,
a size-dependent free
from
the
only
urea
resulted
interaction The
interaction. in
the
use
between as
irreversible
eluent
solute of
phosphate
adsorption
of
57
humic
acids
onto
phosphate
and
When shows
a buffer that
case
of
excluded
is
molecules
the
humic
within
solution
of
used
repulsion
at
pH
in
these
seem
to
the sample
pH
7 containing
eluent of
be
between
7.
overcome
by
the
column
pH 4.7,
At
pH
of
volume.
concentration
the
use
of
to
be
of
of
the
aggregation
the
the
were elution
eluent
likely
humic repulsion
eluted
from
pattern
Therefore
best
the
sample
and
samples
shown).
in
most
amount
a portion 7,
pattern
than
molecules
entire
not
elution
larger
the
Further,
(data seems
is
humic
At
and
the
acid
Aggregation
minimized,
urea
be
(Fig.la.),
humic
11.2).
conditions.
total
of
as size
9.2,
can
2 M urea.
a for
was
buffer
gel
perme-
chromatography. We also
acid
was
large
evaluated
column
urea 1 and
washed
elution
into
preparative
pH
this
fractionated
containing at
is
(pH than
occur
molecules
column
ation
Charge
greater
independent
pH
molecular
buffers
may
problem
containing
high
apparent
these
This
buffer
pH.
in
materials.
of
the low
occurs
of
gel
borate
as
of
Sephadex These
eluent. with
system
4 pooled
water,
then
a
with
fractions G-75 fractions redissolved
rechromatography. (namely
using
A,
neutral were
Soil B,
phosphate
recovered in
I
I 100 “0
Elution
by
Fig. 1. Gel permeation (a) Effect of pH on the a rechromatogram of the
chromatography chromatogram fraction of
a
buffer
and
L7y-C
I 150
, ml
on
precipitation
C
D
I
100
“t
volume
humic
D)
buffer
phosphate-urea
A6
5p
C and
I
150
t “t
Elution of humic acid using of SA-1. (b) Fractionation KP-1.
volume, Sephadex of
ml G-75. KP-1 and
58
reapplied
to
As
the
shown
in
excluded
fraction
portion
apparently
same
analytical Fig.
position
as
retarded
or
adsorbed
the
of the
the
gel
pores to
of
buffer
between
gel
In
solution
it
was
increase
in
As
in
standing
before
molecules
in
size
humic
acid
eluted
differences. minimize
amount
be
the
gel
penetration
entirely
recommend of
of
adsorption by
be
we
the
the
would
the
can
Therefore,
to
at
that
solely
separation
excluded
Because
all
humic
is
directly
for
excluded 2,
dissolved into
a long
portion the
became
excluded
a new
tendency
in
the
gel
same
bed.
before
fraction
fraction This
in the
time
Especially
excluded
macromolecules.
is
injected
standing
Fig.
acid
the
use
interaction
solution When
as
the
injection,
the
sample
however,
an
problem. increased
the
case
seemed
to
associate
was
also
found
with of
pH
the
4.7,
in
time
of
large
with
small
humic ones
to
chromatography
with
a
b
d d t
59
100
VO
Elution Fig. (a)
2. Effect pH 4.7. (b)
of pH
volume,
ml
standing 7.0.
time
Elution on
gel
permeation
150 4
volume,
chromatography
the
were
eluent.
onto
is
the
prove
this
the
eluted
fraction
results of
in
molecules
each
eluent
molecule and
urea
humic
use
the
B, were
material,
the
in
material,
injection. the
the
before. contained
fraction
fractions
These by
as
B is
of
gel
way
solute.
left the
shown
the
same
fraction
other
If the
overcome
containing
procedure, then
the
run. of
molecules
gel
molecular
and
this
eluent.
be urea
the
of
rechromatography.
material, of
attributed neutral
the
the of
re-chromatogram
All
sites
can of
exactly portion
the
previous
in
interactions
into
in
adsorption
adsorption
sites
form
So,
in
the
in
leading
increased.
on
the
the
A.
adsorbed
solute
column
lb,
ml of
SA-1.
59
phosphate
buffer
increase
in
greater
than
It
should
different
of
the at
pH
be
noted
before
clarify
this
or
as
with
increase
was
with
the of
in
injection
neutral
chromatogram Fraction
the of
B was
increased
the
gel
permeation
standing
even
in
solution Fig.
bed. fraction
was with
standing
in
was
left
and
hardly
that changed
a part
of
buffer
solution.
-----
after the
the
for except
chromatogram
Humic
before
fraction
a long
time
and
was
permeation
Each
for
excluded
acid
gel
lb.
the
the
gel
preparative
Fig.
the
increased
solution.
standing
3 shows
almost
fraction However,
C by in
To 6 N HCl, After
were
excluded
buffer
A to shown
extremely
solution.
chromatograms
solution.
as
was
chromatography.
disbefore
fraction
B,
after
the
standing.
fraction,
a
which
b
:!
5min 20 hr
-----
5 min
20 hr
Fraction A
t
the was
ethanol-benzene,
:
d d
eluent,
standing
buffer
disappeared
fractions manner
in with
the
buffer
after pooled
gel but
fraction
same
gel
each
in
sample
treated
the
time
contaminated
during
as
of
chromatogram
the
acid,
excluded
buffer
into
buffer time
was
by
humic
and
the
of
acid
treatments,
into in
alkaline
increasing
permeation
standing
followed
acid
before
gel
humic
standing
fractionated
solved
after
the
chromatography
with
the
original
treatment
unchanged
then
that
5 N NaOH,
ethanol-benzene
with
fraction
7.
and
or
same
Moreover,
7.
phenomenon,
1 N HzS04.
alkaline
pH
excluded
Fraction
i
2
I
50
I
100
I
+
“0
Elution Effect Fig. 3. (a) Fraction A, mixed with B or
“t
volume,
I
50
“t
“0
ml
of standing time on gel permeation B and C which were fractionated C.
100
Elution
in
volume,
chromatography Fig. lb. (b)
ml of KP-1. Fraction A
60
Next,
excluded
stand
for
20
fraction
hrs
fraction
increased
directly
injected
assumed
that
and
the
retarded
of
B or
cases,
From
molecular
molecular
to
To
clarify
the
acid
was
acid
in
phosphate
urea
in
the
phosphate
ed
in
linearly
of
relative
the it
acids
to
excluded
to
results
humic
form
was
slowly
micelle-like
However, agreed
closely
with
IO).
The
large
molecules.
small
ones
buffer
B,
to
case
point C and
those
surface
aggregates
As
active
agent.
for
large
humic
of acid
in
of
sample
4,
only
surface became
of
molecules on
These
acids
acid
by
is
for
salt
and
neutral concenthe
exclud-
due
associate a
long
at
about
0.05%. results
Hayano
mainly
may
standing
humic
constant
concentration
humic
of of
tension
activity.
humic
aggregates
Fig.
and
marine of
in
the
surface
property
humic
The
at
low
tension tension
increase
shown
concentration observed
obtained
micelle-like
of an
KP-1.
D showed
Consequently, form
with
of
surface
surface concentration
decreased
was
active
increasing tension
a surface
the The
surface
increasing
bending
fractions
urea the
formation, method.
with
The
as with
The
micelle Wilhelmy
decreased
containing
A behaved
dyn/cm.
the
solution.
especially
decreased
of by
buffer
buffer
fraction
(ref.
possibility
measured
buffer
tration,
et
al.
to
the
with time
in
solution. Therefore,
to
measure
accurate
molecular
sizes
by
gel
permeation
Fraction
1 0
I 0.05 Concentration
Fig.
allowed
amount
these
size
size
C and
the
decreased
3b).
large
small
fraction both
fraction (Fig.
solution
those
with In
fraction
buffer
with
mixed
solution.
macromolecules.
humic
40
A was
buffer
mixed
in
associated or
in
4.
Surface
tension
of
fraction
I 0.1
1 0.15
of humic of
KP-1.
acid
1 0.2
( % )
chroma-
61
tography
using
emphasized: eluent,
it
To
make
sure
large
soil
humic
They
were
humic
acid
injected
tension
We employed humic
as
eluent,
in
the
into
acid
divided each
the same
the
gel
following
must
solution
bed
low
based
five
and
as
be
the
eluted.
at
low
concentration
of soils.
surface
Group
IV
is
of
related
various
types
acid
on
brown
forest
procedure.
humic
acid
of
them.
In
and
each
case,
concentration,
but
another. method
The
means
Fig.
5.
using
cluster patterns
in
the
on
this
of
surface
and
0.06%.
Almost
Group
II
these
humic
characterized
two
is
a
to
of
humic
were
of
or these
bending
classify
decrease the
acids
calculated
characterised
bending
no
acids
by
of
tension
I
all
showed
analysis
analysis,
Group
concentrations
about
All
tension.
muck,
similarity
in
activity
of
chromatography
IHSS
increasing to
surface
tension
the
Based
shown
from
soils.
on
groups.
are
alluvial
permeation
taxonomic
concentration.
even
gel
to
extract
vs.
have
surface
calcarious,
with
one
indeed
the
according
groups
tension
derived
volcanic,
decreased
and
acids
performed
purified
from
into class
humic measured
a numerical
tension
surface
also from
varied
surface
soil we
and
soils, surface
high
the
and
pattern
ash
that
acids
the
the
immediately
molecules,
the
for
be
extracted
alluvial
were
system
of
2.
the
the
buffer-urea
dissolution
must
Experiment
to
phosphate after
by crimped humic
point
acid
and
were
derived
from
bending
points
and
low
point
at were
relatively volcanic consists
of
b
.E z :: If
L
d I
Soil
II
III
group
Cluster Fig. 5. tension of various five soil groups.
WV
40
' 0.01
I 0.02
I
Concentration
II11111 0.05
0.1
of
analysis classifying the decreasing soil humic acids. (a) Dendrogram (b) Means for surface tension of
humic pattern showing the five
1 0.2
--.Lw
11111111 0.5
acid
1.0
(%)
of surface the relation groups.
of
62 the
humic
bending
acids
surfactant the
extracted
points
were solution
adsorption
Group ance
of
for
a
a minimum
is
in
excluded
fraction
surface
tension.
Thus
showed
higher
it
surface
the
of As
weight of
and
these
humic in
IV,
which
is
characteristic
was
confirmed
activity
acids
Fig.
6,
of
and
was
low
reported
run
on
Sephadex
excluded surface
II.
group
that
humic
will
associate
to
the
acid
G-75
fraction tension.
which
acids
11).
appear-
12).
a large
showed
(ref. The
was
showed
with
large
make
high molecular
micelle-like
aggregates.
In
order
cant by
to
estimate
differences analysis
indicated
in of
in
the mean
values
variance. Table
2.
differences
If
among
for
humic
physico-chemical
The
mean
values
no
significant
groups,
properties
for
each
difference
of
the
was
signifiwere
properties detected
assessed are
between
b
Avo Elul~on
Fig. (a)
6. group
Gel
I,
A4 rolumc
permeation (b) group
A
Elul~on
II
chromatography and (c)
A
vo
of group
VI
Elutlon
rolume
IV
soil by
humic cluster
a
by
polymer 0.06%.
(ref.
of
explained
curve
complexes
seen
was
molecular
Fe(I1)
added
two curve
concentration
tension-concentration
with
I
Similarly,
This
high
at
surface
groups
A small
size
the
minimum
buffer. of
soils.
tension-concentration
polyvinylpyrolidone.
chromatography
characteristic
forest
surface
onto
the
urea-phosphate
brown
the
added
solution
permeation
acid in
surfactant
a distinct
surfactant
Gel using
with
of
V showed
from
reported
acids classified analysis.
volume
into
63 Table
Means
2.
for
physico-chemical
X-3-X
t** 1% &oonm
surface tension (0.15%)
Group
properties
t
Alog
of
soil
groups.
*
IR 2900-l
K
coo;
C=S
(me/g)
ocHyH*
(me/g)
I
49.5(c)
Zl.l(b,c)
0.681(a)
0.117(a)
3.28(b)
2.13(b)
133(a)
II III IV
61.5(a)
67.7(a)
0.500(b)
0.058(b)
4.88(a)
4.47(a)
29(b)
53.8(b)
33.9(b)
0.591(a.b)
O.O97(a,b)
3.68(b)
3.17(a.b)
49.0(c)
12.5(c)
0.665(a)
0.112(a)
3.56(b)
2.48(a)
V
53.8(b)
30.9(b)
0.551(a.b)
0.127(a)
3.53(b)
3.15(a.b)
** Group
ic9-z
W-H
R-H
I
44.7(a)
61.6(b)
II III
19.5(b)
25.3(c)
47.5(a)
63.8(b)
IV
69.0(a)
v
53.5(a)
w C-aY
C-c:
24.6(a)
95.6(b) 45.6(c)
8.4(b)
fa
Hau/Cz
55.1(b)
0.58(b)
0.80(a)
123.4(a)
0.69(a)
0.63(b)
22.8(a)
94.5(b)
66.3(b)
0.58(a)
0.77(a.b)
107.4(a)
34.4(a)
132.0(a)
16.6(b)
0.48(c)
0.92(a)
63.2(b)
22.3(a)
103.5(a,b)
62.6(b)
0.56(b)
0.78(a,b)
aromatic ring carbons: ratio of aromatic carbon ratio of the presumed Ladner
(1960).
were
used
C-(H,+H~+H1,,,)/2-(Hy+HOcH3)/3-CCOOH-Cc=o
=
C
H,,tH,/z+CcooHtCc=o+H0oHtHocH3/3 C-(Ha+H@lact)/2(Hy+HocH3)/3-CcooH-Cc=o
Hau -= Ca
groups
at
the
brackets. humic
acid lower
OCH3
these
than at
for
the
I
IV,
which
groups
of
cm-',
and
spectra.
and
those
2900
terminal
carbon from
and
were
have And
content
of
side-chain this
high
by
of
modified
symbol at
of
8-proton,
group
II
is
which
by ring
from
were
nm of signifiby
very
were
aromatic
in 600
characterized and
characterized
bridge
same
a lot
COOH groups,
is
parameters
the
coefficient
protons,
group
content
structural
coded
1% extinction
II.
group high
Further,
aliphatic
groups
values
groups
estimated (ref.
level,
mean
in
absorption
lH-NMR
5%
The
cantly
of
99(a)
WX
ter-H
C-aro: non-bridge C-ali: aliphatic substituent; C-car: bridge aromatic ring carbons; fa: molar to total carbon: Hau/Ca: atomic hydrogen/carbon unsubstituted aromatic structure. The following formulae, modified from Brown and for the computation. fa
99(a) 138(a)
low
estimated its
low
from content
carbon,
Brown
and
small
content
of
which
Ladner's
were method
13). In
activity
conclusion, are
the constituted
results of
suggest hardly
that condensed
humic
acids
aromatic
showing rings
a high as
the
surface
nucleus
64
and
have
the
humic
many
aromatic groups
acids rings
and
low
8-protons
and
relatively
low
surface
nucleus
and
showing as
the
content
of
OCH3
long activity
side are
characterized
groups
and
chains.
by side-chain
On the
made
up
high
of content
terminal
other
well
hand,
condensed of
COOH
protons.
REFERENCES
4
5 6 7 8 9 10 11
12
13
G. Ferrari and G. Dell'agnola, Fractionation of the organic matter of soil by gel filtration through sephadex, Soil Sci., 96 (1963) 418-421. I. Lindqvist, Adsorption effects in gel filtration of humic acid, Acta Chem. Stand. 21 (1967) 2564-2566 R. S. Swift, B. K. Thornton and A. M. Posner, Spectral characteristics of a humic acid fractionated with respect to molecular weight using an agar gel, Soil Sci., 110 (1970) 93-99 A. M. Posner, Importance of electrolyte in the determination of molecular weights by 'Sephadex' gel filtration, with especial reference to humic acid, Nature, 198 (1963) 1161-1163 R. S. Swift and A. M. Posner, Gel chromatography of humic acid, J. Soil Sci., 22 (1971) 237-249 K. Yonebayashi and T. Hattori. Studies on gel-chromatography of humic 48 (1977) 130-136 (in Japanese) acid, J. Sci. Soil Manure, Jpn., Y. Chen and M. Schnitzer, The surface tension of aqueous solutions of soil humic substances, Soil Sci., 125 (1978) 7-15 M. Tschapek and C. Wasowki, The surface activity of humic acid, Geochim. Cosmochim. Acta, 40 (1976) 1343-1345 A. A. Ray (Ed.), SAS user's guide: Statistic W. S. Sarle, Clustering, in: 1982 ed., SAS institute, Cary, NC, USA, 1982, pp. 417-461 S. Hayano, N. Shinozuka and M. Hyakutake, Surface active properties of marine humic acids, Yukagaku (Oil Chem.), 31 (1982) 357-362 H. Arai, M. Murata and K. Shinoda, The interaction between polymer and surfactant: The composition of the complex between polyvinylpyrrolidone and sodium alkyl sulfate as revealed by surface tension, dialysis, and solubilization, J. Colloid Interface Sci., 37 (1971) 223-227 S. Ozeki, S. Tachiyashiki, S. Ikeda, and H. Yamatera, The interaction of anionic surfactants with an Fe(I1) chelate, J. Colloid Interface Sci., 91 (1983) 430-438 K. Yonebayashi, in preparation.
SURFACE
K.
of the Total Environment,
Science
Publishers
ACTIVE
B.V.,
PROPERTIES
YONEBAYASHI
and
T.
62 (1987) 55-64
Amsterdam
OF
- Printed
SOIL
HUMIC
55
in The Netherlands
ACIDS
HATTORI
Faculty of Agriculture, 606 kyoto (Japan)
Kyoto
Prefectural
University,
Shimogamo,
Sakyo-ku,
SUMMARY For gel permeation chromatography of humic acids, use of a neutral phosIt was determined that the phate buffer containing 2 M urea is recommended. excluded fraction increased with an increase in standing time of sample in buffer solution before injection into the column. From the gel permeation chromatography of mixed samples of excluded and retarded fractions and meait was concluded that the surement of the surface tension of each fraction, reason is that large humic molecules tend to associate with small ones to form micelle-like aggregates. Various types of humic acids were subjected to the measurement of surface tension and gel permeation chromatography. Humic acid containing a large amount of excluded fraction showed high surface activity. The relationships between surface activity and structural properties of humic acid are discussed.
INTRODUCTION Gel
permeation
humic
chromatography
materials
between
(refs.
humic
static
substances
and
interactions We have
and
alkaline
In
this
surface
MATERIALS Soil
an of
samples
and
humic soil
to
applied
have
to
overcome
such
as
the
studies
of
interactions
adsorption
and
electro-
gel
can
6).
In
acids
be
to
Sephadex
gel
by
use
eliminated
practice.
the
however,
of
alkaline
materials. buffer
acid
of
humic
behavior (ref.
appropriate humic
of
in
humic
solution
this
was
buffer
acid
(refs.
examined
solution
was
as
eluent.
related
to
the
7.8).
METHODS
Thirty-five selected
extensively
problems
for
properties
AND
materials,
eluent
undesirable
active
gel
adsorption as
research,
researchers
4,5).
that
association
been
and
adsorption
solution
are
the
the
found
urea
conditions
and
(refs.
studied
materials
has
l-3),
include
types
of
soil
and
Table
1.
Of
these
Humic
acids
IHSS
procedure.
were
0048-9697/87/$03.50
acids
samples
were
used
samples
that
differed
land
use.
samples, extracted
0 1987
A brief only from
Elsevier
as
dried
in
SA-1
these
soils
soils.
degree
description
two,
Science
air
of and and
Publishers
KP-1. purified
B.V.
These
of
humification
the
samples
were
used according
soils
were and
is in
given
in
Experiment to
the
1.
56
Table
1.
Soil
Classification
of
classification
soil
Soil
Andosol
samples
No.
(land
soil
SA-2(u,
JP),
SA-3(p,
JP),
TG-3(u,
JP),
MZ-1-l(v,
JP),
or
Muck
or
Brown
Jahgaru Peat
forest
and
THAI),
GF-4(b,
JP),
SG-3(p,
JP),
soil
NM-E(f,
JP),
KPF-l(f,JP),
U:
For
the
gel
JP),
was
M phosphate
between
4.7
into
borate
11.2
JP) JP),
B-l(f,
SG-2(p,
JP),
KP-l(p,
SA-4(p,
JP),
MGV-3(v,
v: virgin, Brazil.
b:
KPF-3(f,
JP),
B-lO(v,
BZ)
BZ), JP),
KP-2(p,JP),
JP)
buried.
the
pH
were
in
the
column.
An
effluent
tension
solution
(pH
of 7)
column
(2
containing
effects
prepared
surface
Sephadex
a glass
buffers
when
were
into
buffer
paddy, BZ:
THAI),
chromatography,
packed or
-
(0.2%)
injected The
upland, p: Thailand,
SG-4(b, KPF-E(f,
TJ-4(f.
JP), JP),
SG-l(p,
permeation
and
tions
JP)
procedure
material, 0.1
MZ-1-9(b,
THAI)
soil
THAI:
Measurement
T-209(p, JP)
soil
TG-l(p.JP),
JP).
Mongol)
ON-2(p,
Gley
forest, Japan,
MOGL(v,
JP),
NG-5(p, f: JP:
JP),
ON-l(v,
KPF-4(f, Grey
MZ-1-6(b,
JP).
Argentina) USA),
PS-16(u,
Mahji
location)
JP),
WU,
Grumusol
and
SA-l(f,
sus-l(u,
Chestnut
use
TG-P(v, MZ-1-3(b, Chernozem
used.
humic
acid
containing
was
cm).
2 M urea, being
same
G-75 X 45
whose
solution
as (400
solutions
the
eluent
nm)
was in
measured
by
gel
solu-
immediately as
0.1
varied
acid
and used
the
were
were
Humic
dissolved was
as eluents
pHs
investigated.
monitor
2 M urea
used The
detector.
M phosphate Wilhelmy
method. Data
processing Cluster
analysis
the
similarity
as
a function
the
SAS
Kyoto
was in
of
RESULTS
Analysis (ref.
AND
Experiment
adopted
pattern
to of
concentration.
(Statistical
University
For
the
classify decrease We used
System)
the in
at
soils
used
surface
the
computer
the
Data
on
tension
the of
program Processing
basis humic
of acid
contained Center
in of
9).
DISCUSSIONS 1
gel
and
gel
and
borate
must
permeation be buffers
chromatography,
a size-dependent free
from
the
only
urea
resulted
interaction The
interaction. in
the
use
between as
irreversible
eluent
solute of
phosphate
adsorption
of
57
humic
acids
onto
phosphate
and
When shows
a buffer that
case
of
excluded
is
molecules
the
humic
within
solution
of
used
repulsion
at
pH
in
these
seem
to
the sample
pH
7 containing
eluent of
be
between
7.
overcome
by
the
column
pH 4.7,
At
pH
of
volume.
concentration
the
use
of
to
be
of
of
the
aggregation
the
the
were elution
eluent
likely
humic repulsion
eluted
from
pattern
Therefore
best
the
sample
and
samples
shown).
in
most
amount
a portion 7,
pattern
than
molecules
entire
not
elution
larger
the
Further,
(data seems
is
humic
At
and
the
acid
Aggregation
minimized,
urea
be
(Fig.la.),
humic
11.2).
conditions.
total
of
as size
9.2,
can
2 M urea.
a for
was
buffer
gel
perme-
chromatography. We also
acid
was
large
evaluated
column
urea 1 and
washed
elution
into
preparative
pH
this
fractionated
containing at
is
(pH than
occur
molecules
column
ation
Charge
greater
independent
pH
molecular
buffers
may
problem
containing
high
apparent
these
This
buffer
pH.
in
materials.
of
the low
occurs
of
gel
borate
as
of
Sephadex These
eluent. with
system
4 pooled
water,
then
a
with
fractions G-75 fractions redissolved
rechromatography. (namely
using
A,
neutral were
Soil B,
phosphate
recovered in
I
I 100 “0
Elution
by
Fig. 1. Gel permeation (a) Effect of pH on the a rechromatogram of the
chromatography chromatogram fraction of
a
buffer
and
L7y-C
I 150
, ml
on
precipitation
C
D
I
100
“t
volume
humic
D)
buffer
phosphate-urea
A6
5p
C and
I
150
t “t
Elution of humic acid using of SA-1. (b) Fractionation KP-1.
volume, Sephadex of
ml G-75. KP-1 and
58
reapplied
to
As
the
shown
in
excluded
fraction
portion
apparently
same
analytical Fig.
position
as
retarded
or
adsorbed
the
of the
the
gel
pores to
of
buffer
between
gel
In
solution
it
was
increase
in
As
in
standing
before
molecules
in
size
humic
acid
eluted
differences. minimize
amount
be
the
gel
penetration
entirely
recommend of
of
adsorption by
be
we
the
the
would
the
can
Therefore,
to
at
that
solely
separation
excluded
Because
all
humic
is
directly
for
excluded 2,
dissolved into
a long
portion the
became
excluded
a new
tendency
in
the
gel
same
bed.
before
fraction
fraction This
in the
time
Especially
excluded
macromolecules.
is
injected
standing
Fig.
acid
the
use
interaction
solution When
as
the
injection,
the
sample
however,
an
problem. increased
the
case
seemed
to
associate
was
also
found
with of
pH
the
4.7,
in
time
of
large
with
small
humic ones
to
chromatography
with
a
b
d d t
59
100
VO
Elution Fig. (a)
2. Effect pH 4.7. (b)
of pH
volume,
ml
standing 7.0.
time
Elution on
gel
permeation
150 4
volume,
chromatography
the
were
eluent.
onto
is
the
prove
this
the
eluted
fraction
results of
in
molecules
each
eluent
molecule and
urea
humic
use
the
B, were
material,
the
in
material,
injection. the
the
before. contained
fraction
fractions
These by
as
B is
of
gel
way
solute.
left the
shown
the
same
fraction
other
If the
overcome
containing
procedure, then
the
run. of
molecules
gel
molecular
and
this
eluent.
be urea
the
of
rechromatography.
material, of
attributed neutral
the
the of
re-chromatogram
All
sites
can of
exactly portion
the
previous
in
interactions
into
in
adsorption
adsorption
sites
form
So,
in
the
in
leading
increased.
on
the
the
A.
adsorbed
solute
column
lb,
ml of
SA-1.
59
phosphate
buffer
increase
in
greater
than
It
should
different
of
the at
pH
be
noted
before
clarify
this
or
as
with
increase
was
with
the of
in
injection
neutral
chromatogram Fraction
the of
B was
increased
the
gel
permeation
standing
even
in
solution Fig.
bed. fraction
was with
standing
in
was
left
and
hardly
that changed
a part
of
buffer
solution.
-----
after the
the
for except
chromatogram
Humic
before
fraction
a long
time
and
was
permeation
Each
for
excluded
acid
gel
lb.
the
the
gel
preparative
Fig.
the
increased
solution.
standing
3 shows
almost
fraction However,
C by in
To 6 N HCl, After
were
excluded
buffer
A to shown
extremely
solution.
chromatograms
solution.
as
was
chromatography.
disbefore
fraction
B,
after
the
standing.
fraction,
a
which
b
:!
5min 20 hr
-----
5 min
20 hr
Fraction A
t
the was
ethanol-benzene,
:
d d
eluent,
standing
buffer
disappeared
fractions manner
in with
the
buffer
after pooled
gel but
fraction
same
gel
each
in
sample
treated
the
time
contaminated
during
as
of
chromatogram
the
acid,
excluded
buffer
into
buffer time
was
by
humic
and
the
of
acid
treatments,
into in
alkaline
increasing
permeation
standing
followed
acid
before
gel
humic
standing
fractionated
solved
after
the
chromatography
with
the
original
treatment
unchanged
then
that
5 N NaOH,
ethanol-benzene
with
fraction
7.
and
or
same
Moreover,
7.
phenomenon,
1 N HzS04.
alkaline
pH
excluded
Fraction
i
2
I
50
I
100
I
+
“0
Elution Effect Fig. 3. (a) Fraction A, mixed with B or
“t
volume,
I
50
“t
“0
ml
of standing time on gel permeation B and C which were fractionated C.
100
Elution
in
volume,
chromatography Fig. lb. (b)
ml of KP-1. Fraction A
60
Next,
excluded
stand
for
20
fraction
hrs
fraction
increased
directly
injected
assumed
that
and
the
retarded
of
B or
cases,
From
molecular
molecular
to
To
clarify
the
acid
was
acid
in
phosphate
urea
in
the
phosphate
ed
in
linearly
of
relative
the it
acids
to
excluded
to
results
humic
form
was
slowly
micelle-like
However, agreed
closely
with
IO).
The
large
molecules.
small
ones
buffer
B,
to
case
point C and
those
surface
aggregates
As
active
agent.
for
large
humic
of acid
in
of
sample
4,
only
surface became
of
molecules on
These
acids
acid
by
is
for
salt
and
neutral concenthe
exclud-
due
associate a
long
at
about
0.05%. results
Hayano
mainly
may
standing
humic
constant
concentration
humic
of of
tension
activity.
humic
aggregates
Fig.
and
marine of
in
the
surface
property
humic
The
at
low
tension tension
increase
shown
concentration observed
obtained
micelle-like
of an
KP-1.
D showed
Consequently, form
with
of
surface
surface concentration
decreased
was
active
increasing tension
a surface
the The
surface
increasing
bending
fractions
urea the
formation, method.
with
The
as with
The
micelle Wilhelmy
decreased
containing
A behaved
dyn/cm.
the
solution.
especially
decreased
of by
buffer
buffer
fraction
(ref.
possibility
measured
buffer
tration,
et
al.
to
the
with time
in
solution. Therefore,
to
measure
accurate
molecular
sizes
by
gel
permeation
Fraction
1 0
I 0.05 Concentration
Fig.
allowed
amount
these
size
size
C and
the
decreased
3b).
large
small
fraction both
fraction (Fig.
solution
those
with In
fraction
buffer
with
mixed
solution.
macromolecules.
humic
40
A was
buffer
mixed
in
associated or
in
4.
Surface
tension
of
fraction
I 0.1
1 0.15
of humic of
KP-1.
acid
1 0.2
( % )
chroma-
61
tography
using
emphasized: eluent,
it
To
make
sure
large
soil
humic
They
were
humic
acid
injected
tension
We employed humic
as
eluent,
in
the
into
acid
divided each
the same
the
gel
following
must
solution
bed
low
based
five
and
as
be
the
eluted.
at
low
concentration
of soils.
surface
Group
IV
is
of
related
various
types
acid
on
brown
forest
procedure.
humic
acid
of
them.
In
and
each
case,
concentration,
but
another. method
The
means
Fig.
5.
using
cluster patterns
in
the
on
this
of
surface
and
0.06%.
Almost
Group
II
these
humic
characterized
two
is
a
to
of
humic
were
of
or these
bending
classify
decrease the
acids
calculated
characterised
bending
no
acids
by
of
tension
I
all
showed
analysis
analysis,
Group
concentrations
about
All
tension.
muck,
similarity
in
activity
of
chromatography
IHSS
increasing to
surface
tension
the
Based
shown
from
soils.
on
groups.
are
alluvial
permeation
taxonomic
concentration.
even
gel
to
extract
vs.
have
surface
calcarious,
with
one
indeed
the
according
groups
tension
derived
volcanic,
decreased
and
acids
performed
purified
from
into class
humic measured
a numerical
tension
surface
also from
varied
surface
soil we
and
soils, surface
high
the
and
pattern
ash
that
acids
the
the
immediately
molecules,
the
for
be
extracted
alluvial
were
system
of
2.
the
the
buffer-urea
dissolution
must
Experiment
to
phosphate after
by crimped humic
point
acid
and
were
derived
from
bending
points
and
low
point
at were
relatively volcanic consists
of
b
.E z :: If
L
d I
Soil
II
III
group
Cluster Fig. 5. tension of various five soil groups.
WV
40
' 0.01
I 0.02
I
Concentration
II11111 0.05
0.1
of
analysis classifying the decreasing soil humic acids. (a) Dendrogram (b) Means for surface tension of
humic pattern showing the five
1 0.2
--.Lw
11111111 0.5
acid
1.0
(%)
of surface the relation groups.
of
62 the
humic
bending
acids
surfactant the
extracted
points
were solution
adsorption
Group ance
of
for
a
a minimum
is
in
excluded
fraction
surface
tension.
Thus
showed
higher
it
surface
the
of As
weight of
and
these
humic in
IV,
which
is
characteristic
was
confirmed
activity
acids
Fig.
6,
of
and
was
low
reported
run
on
Sephadex
excluded surface
II.
group
that
humic
will
associate
to
the
acid
G-75
fraction tension.
which
acids
11).
appear-
12).
a large
showed
(ref. The
was
showed
with
large
make
high molecular
micelle-like
aggregates.
In
order
cant by
to
estimate
differences analysis
indicated
in of
in
the mean
values
variance. Table
2.
differences
If
among
for
humic
physico-chemical
The
mean
values
no
significant
groups,
properties
for
each
difference
of
the
was
signifiwere
properties detected
assessed are
between
b
Avo Elul~on
Fig. (a)
6. group
Gel
I,
A4 rolumc
permeation (b) group
A
Elul~on
II
chromatography and (c)
A
vo
of group
VI
Elutlon
rolume
IV
soil by
humic cluster
a
by
polymer 0.06%.
(ref.
of
explained
curve
complexes
seen
was
molecular
Fe(I1)
added
two curve
concentration
tension-concentration
with
I
Similarly,
This
high
at
surface
groups
A small
size
the
minimum
buffer. of
soils.
tension-concentration
polyvinylpyrolidone.
chromatography
characteristic
forest
surface
onto
the
urea-phosphate
brown
the
added
solution
permeation
acid in
surfactant
a distinct
surfactant
Gel using
with
of
V showed
from
reported
acids classified analysis.
volume
into
63 Table
Means
2.
for
physico-chemical
X-3-X
t** 1% &oonm
surface tension (0.15%)
Group
properties
t
Alog
of
soil
groups.
*
IR 2900-l
K
coo;
C=S
(me/g)
ocHyH*
(me/g)
I
49.5(c)
Zl.l(b,c)
0.681(a)
0.117(a)
3.28(b)
2.13(b)
133(a)
II III IV
61.5(a)
67.7(a)
0.500(b)
0.058(b)
4.88(a)
4.47(a)
29(b)
53.8(b)
33.9(b)
0.591(a.b)
O.O97(a,b)
3.68(b)
3.17(a.b)
49.0(c)
12.5(c)
0.665(a)
0.112(a)
3.56(b)
2.48(a)
V
53.8(b)
30.9(b)
0.551(a.b)
0.127(a)
3.53(b)
3.15(a.b)
** Group
ic9-z
W-H
R-H
I
44.7(a)
61.6(b)
II III
19.5(b)
25.3(c)
47.5(a)
63.8(b)
IV
69.0(a)
v
53.5(a)
w C-aY
C-c:
24.6(a)
95.6(b) 45.6(c)
8.4(b)
fa
Hau/Cz
55.1(b)
0.58(b)
0.80(a)
123.4(a)
0.69(a)
0.63(b)
22.8(a)
94.5(b)
66.3(b)
0.58(a)
0.77(a.b)
107.4(a)
34.4(a)
132.0(a)
16.6(b)
0.48(c)
0.92(a)
63.2(b)
22.3(a)
103.5(a,b)
62.6(b)
0.56(b)
0.78(a,b)
aromatic ring carbons: ratio of aromatic carbon ratio of the presumed Ladner
(1960).
were
used
C-(H,+H~+H1,,,)/2-(Hy+HOcH3)/3-CCOOH-Cc=o
=
C
H,,tH,/z+CcooHtCc=o+H0oHtHocH3/3 C-(Ha+H@lact)/2(Hy+HocH3)/3-CcooH-Cc=o
Hau -= Ca
groups
at
the
brackets. humic
acid lower
OCH3
these
than at
for
the
I
IV,
which
groups
of
cm-',
and
spectra.
and
those
2900
terminal
carbon from
and
were
have And
content
of
side-chain this
high
by
of
modified
symbol at
of
8-proton,
group
II
is
which
by ring
from
were
nm of signifiby
very
were
aromatic
in 600
characterized and
characterized
bridge
same
a lot
COOH groups,
is
parameters
the
coefficient
protons,
group
content
structural
coded
1% extinction
II.
group high
Further,
aliphatic
groups
values
groups
estimated (ref.
level,
mean
in
absorption
lH-NMR
5%
The
cantly
of
99(a)
WX
ter-H
C-aro: non-bridge C-ali: aliphatic substituent; C-car: bridge aromatic ring carbons; fa: molar to total carbon: Hau/Ca: atomic hydrogen/carbon unsubstituted aromatic structure. The following formulae, modified from Brown and for the computation. fa
99(a) 138(a)
low
estimated its
low
from content
carbon,
Brown
and
small
content
of
which
Ladner's
were method
13). In
activity
conclusion, are
the constituted
results of
suggest hardly
that condensed
humic
acids
aromatic
showing rings
a high as
the
surface
nucleus
64
and
have
the
humic
many
aromatic groups
acids rings
and
low
8-protons
and
relatively
low
surface
nucleus
and
showing as
the
content
of
OCH3
long activity
side are
characterized
groups
and
chains.
by side-chain
On the
made
up
high
of content
terminal
other
well
hand,
condensed of
COOH
protons.
REFERENCES
4
5 6 7 8 9 10 11
12
13
G. Ferrari and G. Dell'agnola, Fractionation of the organic matter of soil by gel filtration through sephadex, Soil Sci., 96 (1963) 418-421. I. Lindqvist, Adsorption effects in gel filtration of humic acid, Acta Chem. Stand. 21 (1967) 2564-2566 R. S. Swift, B. K. Thornton and A. M. Posner, Spectral characteristics of a humic acid fractionated with respect to molecular weight using an agar gel, Soil Sci., 110 (1970) 93-99 A. M. Posner, Importance of electrolyte in the determination of molecular weights by 'Sephadex' gel filtration, with especial reference to humic acid, Nature, 198 (1963) 1161-1163 R. S. Swift and A. M. Posner, Gel chromatography of humic acid, J. Soil Sci., 22 (1971) 237-249 K. Yonebayashi and T. Hattori. Studies on gel-chromatography of humic 48 (1977) 130-136 (in Japanese) acid, J. Sci. Soil Manure, Jpn., Y. Chen and M. Schnitzer, The surface tension of aqueous solutions of soil humic substances, Soil Sci., 125 (1978) 7-15 M. Tschapek and C. Wasowki, The surface activity of humic acid, Geochim. Cosmochim. Acta, 40 (1976) 1343-1345 A. A. Ray (Ed.), SAS user's guide: Statistic W. S. Sarle, Clustering, in: 1982 ed., SAS institute, Cary, NC, USA, 1982, pp. 417-461 S. Hayano, N. Shinozuka and M. Hyakutake, Surface active properties of marine humic acids, Yukagaku (Oil Chem.), 31 (1982) 357-362 H. Arai, M. Murata and K. Shinoda, The interaction between polymer and surfactant: The composition of the complex between polyvinylpyrrolidone and sodium alkyl sulfate as revealed by surface tension, dialysis, and solubilization, J. Colloid Interface Sci., 37 (1971) 223-227 S. Ozeki, S. Tachiyashiki, S. Ikeda, and H. Yamatera, The interaction of anionic surfactants with an Fe(I1) chelate, J. Colloid Interface Sci., 91 (1983) 430-438 K. Yonebayashi, in preparation.