properties of the yellow organic acids of natural waters

PROPERTIES
OF THE YELLOW ORGANIC
NATURAL
WATERS’
ACIDS
OF
Masood Ghassemi and R. F. Christman
Department
of Civil
Engineering,
University
of Washington,
Seattle
98105
ABSTRACT
Gel filtration
profiles obtained from a variety of naturally
colored waters indicate that
color producing molecules are mostly in the apparent molecular weight range of 700-10,000
relative to dextrans.
One fraction seems to have a molecular weight in excess of 50,000.
The apparent size of the color molecules was found to be pH dependent, increasing as pH
increases.
Size increases are attributed
to increased functional
group ionization
and a
decrease in the adsorbability
on Sephadex.
The degree of association of color molecules with iron was also found to bc pH dependent, being greater at both high and low pH than in the range of 7-8.
An understanding
of the organic substances present in a body of water is essential to properly assessing their role in
productivity.
The so-called yellow organic
acids, comprising a broad class of polyhydroxy-methoxy-carboxylic
acids and quiare common
nones of plant
origin,
constituents of almost all surface waters.
Bccausc of their absorption of light cncrgy
and interference with photosynthesis, high
concentrations of color acids may reduce
In lower
concentrations,
productivity.
color acids are considered beneficial by
virtue of their association with metal nutrients. Iron, for example, has been shown
to remain in solution in the prescncc of
color acids even under conditions of high
pH and rcdox potential (Shapiro 1966).
This paper presents some recent findings on the physical and optical properties
of the yellow organic acids and on their
mode of association with iron in the
aquatic environment,
The chemical naturc of these acids and its uniformity
in
different waters was discussed by Christman and Ghasscmi (1966).
graphic and climatic conditions,
The six
waters on which most of the analysts were
performed
are listed in Table 1 with
symbolic designations for referencing convenicncc.
The waters from Alaska were flown to
Seattle in glass bottles. The local waters
and those from British Columbia were
brought to the laboratory in 19-liter polyAll samples were
ethylene containers.
filtered
through
a slow filter
paper
(Eaton-Dikeman
No. 613) and concentrated by vacuum evaporation at a tcmperature of less than 40C. Except that
large amounts of salts, mostly inorganic,
precipitated during concentration from the
Alaska waters, the quantity of precipitate
observed was small. All concentrates were
filtered through 450-rnp Millipore
filters
and stored at 5C. Some samples developed
a precipitate upon long standing which
was removed by filtration,
The authors are indebted to Dr. V. A.
Dugdale and Mr. S. Wishart for their
assistance in obtaining water samples.
Size fractionation
METHOD
OF
STUDY
on Sephadex
Scphadcx gel filtration was used for the
fractionation
of organic color (Table 2).
The beds were prepared in 25- or 28-mmdiam Pyrex tubes, 50 to 60 cm long.
Five ml of the sample to be fractionated
was applied to the top of the bed with a
’ This work was supported by Federal Water
bent-tip pipette.
The elution was perPollution
Control Administration
Research Grant
formed under a slight head using Clark
WF EF 01031.
583
Sample collection and concentration
The waters used came from several
streams in western Washington and from
other locations representing different gco-
584
rrABLE
MASOOD
1.
Sources
Water
M.G.
PI.
P.H.
B.H.
S.L.
V.L.
Source
and designations
waters
and
GIIASSEMI
of
R.
analysis and instrumental
methods
Total iron was measured as follows: An
approximate volume of the sample was
evaporated to dryness in a Kjcldahl flask
and the organic matter destroyed by the
addition of 5 ml of concentrated nitric acid
and 5 ml of 30% hydrogen pcroxidc. The
residue was picked up in 4 N IICl and its
pH raised to about 5 with concentrated
ammonium hydroxide.
The bathophenanthroline procedure as described by Lee
and Stumm (1960) was then followed.
Total organic carbon was determined in
the apparatus described by Christman and
Ghasscmi ( 1966).
All quantitative
fluorescence measurcmcnts were made with a Turner model
CIIRISTMAN
G-10
G-25 ( coarse )
G-50 ( fine )
G-75
G-100
be
* Molecular
separated
of Sephadex gel
Properties
Approx
limit
for
complete
exclusion
(mol wt)
Sephadex
type
location
and Lubs buffers or solutions of 0.01 N
NaCl with pH adjustment made with HCl
or NaOH. Convenient volumes of the eluent leaving the column were collected with
an automatic fraction collector
( Model
1250 series, Warner-Chilcott
Lab. Instr.
Div., Richmond, Calif. ) . Some or all of
the following
determinations
were made
on the collected fractions: carbon content,
absorption at 350 rnp, fluorescence, and
total iron.
Blue Dextran 2000 and Dextran 10 (both
manufactured by Pharmacia Fine Chcm. )
and glucose were used for column calibration. Blue Dextran 2000 has an average
molecular weight of 2,000,OOO and is cxeluded by all types of Sephadcx. The
weight average and number average molccular weights for Dextran 10 were 11,200
and 5,700, respectively.
F.
TABLE 2.
colored
Creek in Skagit Co., Washington
Pilchuck Creek, Skagit Co., Washington
Keogh River, Port Hardy, British Columbia
Earth Impoundment,
Bull Harbour,
British Columbia
Smith Lake, Collcgc, Alaska
Vad Lake, Goldstream Valley, Fairbanks,
Alaska
Chemical
AND
by
weight
size,
Fractionation
range*
(mol wt)
700
5,000
10,000
50,000
100,000
limits
within
with
respect
up to 700
1004,000
50%10,000
l,OOO-50,000
5,000-100,000
to
which
molecules
polysaccharides.
can
110 fluorometer.
The instrument
was
equipped with a primary filter peaking at
360 mp and a secondary filter with a sharp
cut at 415 rnp. All fluorescence spectra
were obtained on Fluorospec model SF-l
( Baird-Atomic, Cambridge, Mass. ) .
Color absorption
measurements were
made at 350 rnp using a Beckman model
DU quartz spcctrophotome ter. The ultraviolet and visible spectra were determined
on a Beckman DK-2A ratio recording spectrophotometer.
A Beckman Zeromatic II
pH meter was used for all pH measurements. Color determinations
were made
with a Lumetron model 402-E photoclectric calorimeter
(Photovolt
Corp., New
York), using a 420-rnp filter. A color standard was prepared in accordance with the
standard procedure of the American Public IIcalth Association ( 1965).
RESULTS
AND
DISCUSSION
Raw water analysis
Partial chemical analyses of waters used
in this study are given in Table 3. The
samples from Alaska (V.L. and S.L. ) were
taken during winter from beneath the ice
cover. This could have increased their
high mineral content. The data in Table
3 show that, although iron is present in
all colored waters, there is no linear relationship between color intensity and iron
content-a
fact noted by several other
investigators.
An interesting property of the yellow
All
organic acids is their fluorescence.
waters investigated showed a broad band
of fluorescence centering between 450 and
460 rnp with the wavelength of maximum
YELLOW
ORGANIC
TABLE 3.
Water
at ‘PI-1 7
at ~11 7
Dissolved organic
carbon, ppm
iron,
ppm
Specific conductance,
pmhos/cm
Total
P.I.
alkalinity,
ppm as CRC03
Hardness,
Ckl*+
Mg”+
Total
IN
NATURAL
585
WATEH
analyses of colored
waters
B.I-I.
P.H.
S.L.
V.L.
7.1
5.4
5.0
6.9
7.4
117
41
237
298
200
260
124
25
85
93
141
372
14
7
18
18
41
69
6.G
Fluorescence
Total
chemical
KG.
sample
PH
Color
Partial
ACIDS
0.11
0.05
0.31
0.28
0.14
1.57
40
24
24
33
182
523
9
11
4
6
48
306
10
4
14
6
6
12
5
1
6
5
3
8
48
22
70
240
140
380
ppm as CaC03
excitation located in the region of 360-370
rnp, The intensities of both color and
fluorescence were affected by pH, although not to the same extent. At low
color values and at a fixed pH, the intensity of fluorescence varied linearly with
Table 3 indicates
color conccn tration.
that the fluorescence-to-carbon
and fluorescence-to-color ratios are not the same
for all waters. This can be cxplaincd in
terms of differences in the chemical nature
of their yellow organic acids or the nonuniformity
of their mineral composition,
or both. In other studies (Ghasscmi and
Christman, unpublished ) , the interaction
of iron and aluminum with yellow organic
acids resulted in the enhancement
or
quenching of the fluorescence, dcpcnding
on the pH and the relative concentrations
of the reacting species.
Sephadex gel filtration
experiments
The size distribution
and its uniformity
among different waters
Several workers have applied Sephadcx
gel filtration to the fractionation of color.
Gjessing ( 1965) worked with the organic
substances of a typical moorland water in
Norway and found two main fractions:
one with a molecular weight between
100,000 and 200,000 and one in the 10,000
range. Shapiro ( 1964) recognized four
distinct elution patterns for the 22 Minncsota lakes he studied. The majority of
organic acids in these lakes were reported
to have a :molecular weight greater than or
close to 10,000. Recent data by Gjessing
and Lee (1967) also indicate that the
yellow organic acids from different waters
may have different elution profiles and
that, in general, low-colored waters have
most of their color in the low molecular
weight fraction; whereas in modcratcly or
highly colored waters, more color is found
in the high molecular weight fraction.
Fig. 1 shows the elution profiles on G-25
Sephadex for the six waters used. With
the possible exception of V.L., all waters
appear to contain the same components,
although in different proportions.
Waters
M.G., B.H., and P.H. contain higher ratios
of larger molecular weight compounds,
while in waters P.I., V.L., and SL., the
smaller molecular weight fractions prcdominate. (In these experiments a buffer
solution was used as eluent to ensure a
constant @H. )
In Fig. 2, the exclusion and inclusion
limits, as determined with a mixture of
Blue Dextran 2000 and glucose, are superimposed on a typical G-25 profile. In this
case, for maximum sensitivity, the fluorescence intensity of the column effluent was
monitored. The data clearly indicate that
although a fraction of organic color was
586
MASOOD
GISASSEMI
AND
(j
F.
CIIRISTMAN
SEPHADEX G-25
ELUENT : pH 6 CIARK AND LUBS BUFFER
BED DIMENSIONS:
2.3CM DIA.
46 CM LONG
0.35
d
R.
0.15
0.10
0.05
0.00
% 0.15
8
;:
4 0.10
d
6
0.05
80
120
100
140
180
160
ELUTION
FIG.
1.
Elution
profiles
200
240
260
280
VOLUME , ml
for six different
cxcludcd from the column most was dctained, with some being reversibly
adsorbed. The G-25 data, thcrcforc, were
not used to make any estimate of the
actual molecular size distributions
except
to compare profiles.
220
colored
waters.
All color molecules were excluded
G-10 Scphadex. With the exception
small fraction that was even cxcludcd
G-75, most of the coloring material
included by G-50.
The Sephaclex data thus indicate
from
of a
from
was
that:
YELLOW
ORGANIC
ACIDS
IN
NATURAL
587
WATER
GLUCOSE
100
ELUENT : pII
CLARK AND LUBS BUFFER
BED DIMENSIONS
: 2.3 CM DIA
46 CM LONG
B
B
s
t:
80
-160
60
-120
E
i
2
k2
=
d
E
ELUTION
FIG.
2.
Adsorption
VOLUME,
a) color molecules are of various sizes and
b ) with respect to dextrans, practically all
color molecules are in the 700 and 10,000
mol wt range with one small fraction cxceeding 50,000. The proportion
of this
SEF’HADEX
ELUENTS:
color
on Sephadex
0.01
N N&l,
BED DIMENSIONS
pH ADJUSTMENT
OR NaOII
:
46
CM LONG
2. 3 CM
DIA
M. G.
:
ELUTION
FIG.
3.
Effect
G-25.
fraction can be estimated on the basis of
its relative area under the profile curve.
Such a figure, however, would not be very
significant unless the pH of the sample is
also specified since, as will be shown,
G-75
BY MCI
WATER
ml
of organic
VOL
\
I
, ml
of pI1 on molecular
size.
MASOOD
GIIASSEMI
AND
R.
F.
CHRISTMAN
SEPHADEX G-50 FINE
ELUENTS : CLARK & LUB BUFFERS
BED DIMENSIONS
: 50 CM LONG
2.3 CM DIA
WATER F’.I.
ELUTION
FIG, 4.
Effect
VOL
, ml
of pH and ionic
composition
the molecular size of the coloring matter,
as determined by Sephadex, is affcctcd
by pH.
The effect of pH on molecular
:
2.3
size.
solutions of 0.01 N NaCl with pH adjustmcnts made by addition of NaOH or HCI.
Fig. 3 shows that the size distribution
of organic color, as determined by Sephadex, is pH dependent (see also Figs. 8
and 9), that is, the molecules appear to
become larger as the pH is raised. To
investigate this phenomenon over a wide
size
To establish the effect of pH on molecular size, elutions were performed with
Clark and Lubs buffer solutions and with
SEPHADEX
G-75
.BED DIMENSIONS
on molecular
CM DIA
46 CM LONG
0.8 -
f?\pI-I
:1.
E
z
c?
7,0
NaOH/I<lI$04
BUFFER
P
0.6-
8,
-
pH 7.0
BORAX/BORIC
ACID
BJFFER
P
P
d
FIG. 5.
Effect
of borate
on the molecular
size distribution
of color.
YELLOW
BLUE DEXTRAN
ORGANIC
ACIDS
IN
NATURAL
2000
P
0.8 -
589
WATER
SEPHADEX
G-50
BED DIMENSIONS
I ’
FINE
: 2.3
CM DIA
50 CM LONG
I\
0.6 -
0.0
50
70
+BLUE
4
I\
0.4
90
DEXTRAN
110
130
150
170
2000
SEPHADEX
G-100
BED DIMENSIONS
: 2.3 CM DIA
40 CM LONG
0.3
0.2
0.1
0.0
40
60
80
EIlJTION
FIG. 6.
Elution
100
VOLUME,
analysis
with
ml
120
0.01
N
140
NaOH.
160
590
MASOOD
GIIASSEMI
AND
R.
F.
CIIRISTMAN
80
60
u
E
2
40
20
0
0.3-L
P\
g 0.6-
0.8
i
BID-E DEXTRAN
2000
I
40
80
I
1
120
I
ELUTION
I
160
--
I
I
200
VOLUME
I
240
, ml
SEPHADEX G- 75
MADE WITH NaOH AND HCl
ELUENTS : 0.01 N NaCl WITH pH ADJUSTMENTS
-o-dpH 3.85,
o--o--opH 5.5, -e-a-o
pH 7.5
FIG.
7.
Effect
of pH on color-carbon-iron
relationships
(water
M.G.).
I
I
280
I
YELLOW
ORGANIC
ACIDS
IN
ELUTION
SEPHADEX G -75
ELUENTS : 0.01 N NaOH
-me
FIG.
7.
NATURAL
WATITR
VOLUME,
AND 0.01 N HC:I
( Continued. )
range of accurately controlled pH levels,
elution profiles were performed with Clark
and Lubs buffer solutions (Fig. 4). The
buffers of pH 4.0 and 5.0 contained NaOII
and potassium acid phthalate. The pH 7.0
ml
-
buffer was made of KIIJ’04
and NaOH.
Buffers of PI-I 8, 9, and 10 contained NaOH
and decreasing amounts of boric acid.
As the pH was increased in the 4.0-7.0
region, there was a slight increase in
MASOOD
50
70
90
GHASSEMI
110
AND
130
150
ELUTION
SEPHADEX
EL.UENTS
IL
F.
CHRISTMAK
170
VOLUME,
210
190
230
ml
G- 75
: 0.01
-h--A
BED DIMENSIONS
FIG.
8.
N NaCl WITH pH ADJUSTMENTS
pH S. 5 AND
- pH 7.5 > -O--Q: 2.3
Effect
CM DIA
AND
MADE
-o-c+
WITH NaOli
pH 3.85
AND
HCl
46 CM LONG
of pH on color-carbon-iron-fluorescence
relationships
(water
P.H.).
250
YELLOW
ORGANIC
ACIDS
molecular size as previously noted. At pH
8.0-10.0, the molecules were much larger
-the majority being excluded from the column. This increase in size, however, is
related to the presence of borate ions as
shown by the gradual decrease in molecu1ar size at pH 9.0 and 10.0 where the
eluents contained less boric acid. This was
also confirmed when elutions were pcrformed at the same pH in the absence and
presence of this acid ( Fig. 5).
The increase in size of organic color
molecules in the presence of boric acid is
understandable,
considering
the polyhydroxy-polycarboxylic
nature of these molecules and the ability of borate ion to form
chelates with such structures.
To establish that the observed increase
in molecular size with increased pH was
not associated with changes in Sephadex
properties, a mixture of Blue Dextran 2000,
Dextran 10, and glucose was fractionated
on a G-75 column using different buffer
solutions. The profiles had identical patterns. Similar results were obtained when
simple phenolic compounds such as quercetin were eluted from a G-75 column at
two different pH values. In the case of
color, however, the extent of adsorption on
Sephadex gel at different pH values could
not bc determined.
When a solution of
0.01 N HCl was used as cluent, the volume
required to elute the sample from the column was greater than that required for
elution of glucose, indicating that under
highly acidic conditions color is reversibly
adsorbed on the gel. If the extent of adsorption of color on Sephadex decreases as
the pH is raised, this may account for the
apparent increase in molecular size of
color with pII. This increase in molecular
size may also be attributed to changes in
molecular shape brought about by an increase in ionization
of the functional
groups at higher pH levels,
As shown in Fig. 6, when a solution of
0.01 N NaOH was used as eluent, the
profile was similar to that obtained with
buffers containing boric acid. One fraction
was excluded by G-100. When elution was
performed with 0.5 M NHdOH, some of
IN
NATURA:L
WATER
593
the color precipitated
at the top of the
bed and could bc washed down by the
addition of strong chelating agents, such
as triethanolamine or Vcrsenex 80.
Iron-color association
The above observations led us to conclude that the large increase in molecular
size at high pH was probably related to
the degree of metal-organic
association,
which woulld vary with changes in the “pH.
In such an association, iron would be the
clcmcnt most likely to be involved because
of its relatively high concentration in the
samples and because of its low solubility at
high PII. Since elucidation of the nature
of iron-color association and its relation to
molecular weight and “pH were additional
objectives of this study, Sephadex experiments were designed to determine the iron,
color, and carbon content of the effluents.
The quantitative recovery of iron was not
possible due to the low precision of the
test and the large number of samples. In
some cases, total iron detected in the cfflucnt was higher than that originally prcscnt
in the sample.
A study of Figs. 7 and 8 reveals that the
cxtcnt of association of iron with color, in
color concentrates with ratios that are
nearly the same as those for the raw
waters, depends on pH. As the pH is
raised from 5.5 to 7.5 (or per,haps to values
not much greater than this), iron appears
to dissociate itself from color. This iron
appeared in the effluent much later than
predicted by the column inclusion limit,
presumably being retarded by adsorption
on the gel. This is especially clear in Fig.
7 and noticeable in Fig. 8.
We prepared a ferric chloride solution
containing the same concentration of iron
as the color concentrate and applied a
sample of it to the column. Practically all
of the iron became irreversibly bound and
could not be eluted. The same result was.
obtained when ferrous sulfate was used
instead of ferric chloride. This indicates,
that the iron in natural waters is chemically bound to organic color or is corn-.
plexed with other chemical species that:
594
MASOOD
GHASSEMI
SEPIIADEX
G-25
ELUL’NT : 0.5% AQUEOUS
SAMPLE
: 1:l DILUTION
AND
SOLIJTION
OF WATER
R.
F.
Or VERSENEX
M. G.
CHRISTMAN
80 (ld i 11)
.0.9
CONCENTRATE
0. a
-0.6
-0.4
E
2
I
-0.3
$
-3.2
-0.1
---o--.c-.-c---o--o--~o-‘-
ELUTION
FIG. 9.
Iron-color
relationship
VOLUME,
-4
ml
in the presence
prevent reaction with the Sephadex. Even
at pH 7.5, iron may still be bound to some
color, the concentration of which is too
small to be detected by the carbon analysis.
Although
the quantitative
relationship
between iron and color as affected by pH
could not be determined, it was established that iron and color can associate to
a greater degree when the pH is slightly
acidic or lower. The dissociation of iron
from color at higher pH values (but not
under very alkaline conditions-see
below )
can be explained in terms of a chelation
model in which hydroxide ions can successfully
compete with
the functional
groups on the colored material for the
coordination sites of iron. This can also
explain why the optimum pH of color
coagulation with ferric chloride is in the
range of 4.5-5.5 for most natural waters.
Some of the iron present in the high molecular weight fractions is probably bound by
a mechanism different from chelation, as
this iron could not be removed even by
strong chelating agents.
Under highly alkaline conditions, soluble
iron-hydroxo complexes become associated
with color again, probably through simple
adsorption. Even at such a high pH, most
of the iron could be pulled away from the
of strong
chelating
agents.
color by a strong chelator. This is indicated in Fig. 9 where a 0.5% solution of
Versenex 80 is used as clucnt. The fraction of iron that is not removed, however,
can still account for the apparent large
molecular size of color at this pH.
The nature of iron-color association has
been extensively invcstigatcd by Shapiro
( 1964). I-Ie reported that the “iron-holding” capacity of color increases with I~>H
up to a value of 10, after which it dccreases rather abruptly.
This finding is
inconsistent with the Scphadcx fractionation data reported here, which indicate
that the color-iron association is greatest
at intermediate
pH values. Shapiro assumed the mode of association to be primarily one of pcptization [i.e., the adsorption of color on Fe ( OH) 3 sols] rather than
chelation. Recent investigations (Ghassemi
and Christman, unpublished), however, indicate that the color-metal ion interaction
proceeds on a purely chemical basis.
Optical properties and molecular sixe
Although there are a few references in
the literature concerning the relation of
optical properties to molecular size, the
conclusions have not been backed by adequate cxperimcntal data.
YELLOW
ORGANIC
ACIDS
0.8
IN
NATURAL
A
C
b
A
A
0.7
595
WATER
A
n
0
CARBON
CONTENT
ABSORBANCE
OF EFF.
ABSORBANCE
IN BASE
ABSORBANCE
IN ACID
SAMPLES
b
a.
0.6
0.5
%
53
;-"
4
d
6
0.4
0.3
0.2
0.1
0.c
ELUTION
SEPH ADEX
ELUENT:
WATER
VOLUME,
ml
G-75
0.01
N
NaCl
M. G.
FIG.
10.
Light
absorption
properties
Fig. 10 shows elution profiles for water
M.G. The optical density, carbon content, and color-to-carbon ratio are plotted
against clution volume. The absorption
data for effluent samples made acidic or
of various
color fractions.
basic by addition of acid or base arc also1
presented; these are designated as A,
and Ah.
The data indicate that a) the optical
density per unit organic carbon content is.
596
MASOOD
0.4
1
HUMIC
GIIASSEMI
AND
R.
F.
CHRISTMAN
C MYMATOMELAh’IC
ACIDS
0.3
0.0
FLU’I‘ION
FIG. 11.
water
Sephadex
G-25
elution
profiles
VOI.UE*Il:
for
, ml
fulvic
and
humic
and hymatomelanic
fractions
of
M.G.
lowest for molecules in the intermediate
size range and highest for large molecular
weight fractions, b) the optical density of
all fractions decreases by addition of acid
and increases with “pII, and c) there appears to be no strict correlation between
the change in absorptivity
with “pII and
molecular size. Some fractions were sclected at random and their fluorescence
and UV spectra determined. The spectra
were identical with those of raw colored
water,
In accordance with the classic solubility
fractionation scheme for soil organic matter, color can be fractionated into humic
acids (soluble in alkali, precipitated
by
acid, and insoluble in alcohol), hymatomelanic acids ( soluble in alkali, precipitated by acid, and soluble in alcohol), and
fulvic acids (soluble in alkali and not precipitated by acid). The fulvic acid fraction has been reported to account for up
to 90% of the weight of color solids. Fig.
11 compares elution profiles of fulvic acid
and humic and hymatomelanic
fractions
and indicates that humic and hymatomelanic acids contain a larger proportion of
high molecular weight compounds than
fulvic acid, This is in agreement with the
findings of Packham (1964) that, per unit
weight, humic acids produce more color
than fulvic acids.
SUMMARY
Sephadex gel filtration has been used to
investigate the molecular size distribution
of the yellow organic acids of natural waters. The six waters studied were obtained
from four streams in western Washington
British Columbia,
and
and Vancouver,
from two lakes in Alaska. Sephadex data
indicate that, with the exception of a fraction having a molecular weight in excess
of 50,000, color molecules are apparently
in the 700-10,000 mol wt range, as comsince the
pared to dextrans. IIowever,
interaction of color acids with the gel could
not be determined, elution profiles obtained on Sephadex cannot be regarded
as true representation
of molecular size
distribution until their validity is confirmed
by other methods of molecular size analysis. Furthermore,
the effect of sample
concentration on molecular size distribution is as yet unknown, and it is entirely
possible that the actual distribution of color
acids in natural waters is quite different
from that indicated by Sephadex.
YELLOW
ORGANIC
ACIDS
Sephadex was also used to study the
effect of pH on molecular size, using
eluents of different pH and ionic composition. In the acidic to slightly alkaline pI1
range, the size of the color molecule
showed a slight increase with increase in
pH. This can be attributed to an increase
in the molecular radius due to an increase
in the ionization of the functional groups,
or a decrease in the extent of adsorption
on Sephadex, or both. Under highly alkaline conditions, the color molecules appear
much larger, presumably due to adsorption
on or complex formation with iron or
other metal-hydroxo complexes. Similarly,
boratcs were found to effect a large increase in molecular
size probably
by
forming
complexes with organic color
molecules.
The degree of association of iron with
color was also studied and found to be
pH dependent, being highest in the lower
pH regions and under highly alkaline conditions, and lowest in the vicinity of pH
7-8. The nature of the iron-color association and its variation with pI1 can bc
cxplaincd in terms of a chelation model
in which the functional groups on color
and the hydroxide ions compete for the
coordination sites of iron, The data, however, contradict
the finding of Shapiro
(1964) that the iron-holding
capacity of
color increases with pH to a maximum
IN
NATURAL
WATER
597
value at pH 10 and then decreases rather
sharply. Sephadex experiments using solutions of strong chelating agents as eluents
indicated that, with the exception of a
small amount of iron bound to large molecular weigh.t fractions, most of the iron
could be pulled away from the color.
REFERENCES
AMERICAN PUBLIC HEXLTFI ASSOCIATION. 1965.
Standard
methods
for the examination
of
water
and wastewater,
12th ed.
APHA,
New York. 592 p.
CIIIILTSTLMAN,R. F., AND M. GHASSEMI. 1966.
Chemical nature of organic color in water.
J. Am. Water Works Assoc., 58: 723-741.
GJESSING,E. T. 1965. Use of sephadex gel for
the estimation of molecular weight of humic
substances in natural waters.
Nature, 208:
1091-1092.
-,
AND G. F. LEE. 1967. Fractionation
of
organic matter in natural waters on sephadex
columns.
Environ.
Sci. Technol.,
1: 631638.
LEE, G. F., AND W. STUMM.
1960. Determination of ferrous iron in the presence of ferric iron with bathophenanthroline.
J. Am.
Water Works ASSOC., 52: 1567-1574.
PACKIIAM, R. F. 1964. Studies of organic color
in natural water.
Proc. Sot. Water Treat.
Exam., 113: 316-334.
SEIAPIRO, J. 1964.
Effect
of yellow
organic
acids on iron and other metals in water.
J.
Am. Water Works Assoc., 56: 1062-1082.
-.
1966.
Yellow
organic
acids of lake
water:
differences
in their composition
and
behavior.
Paper, Symp. Hungarian
Hydrol.
Sot., Budapest-Tihany,
Sept. 25-28, 1966.