Fractionation by molecular weight of organic
substances in Georgia coastal water1
John R. Wheeler
University
of Georgia
Marine
Institute,
Sapelo Island
31327
Abstract
Ultrafiltration
membranes were used to separate organic substances in Georgia coastal
water into fractions to which approximate
molecular weight (MW)
ranges could be assigned. Salt marsh water contained high concentrations
of dissolved organic carbon between
1,000 and 30,000 MW, as well as high concentrations
of dissolved carbohydrate
in all MW
classes. Concentrations
of dissolved carbohydrate
in offshore waters were low, and in some
MW fractions
( especially those >30,009 MW)
carbohydrate
was at times undetectable.
Absorbances at 254 mn by MW fractions roughly paralleled
concentrations
of dissolved
organic carbon. Most of the color of marsh, sound, nearshore, and offshore waters, once
the particles were removed, was due to substances between 1,000 and 30,000 MW.
All organic material in seawater that
passes through a filter with a 0.8-p or
0.45-p pore size has commonly been considered dissolved. Recently attempts have
been made to fractionate what should be
termed filter-passing
(rather
than dissolved) organic substances. Ogura ( 1970)
showed that some organic material in the
East China Sea that passed a 0.45,~ poresize filter was retained by a 0.1-p pore-size
filter, indicating the existence of particles
<0.45 p in diameter. He also suggested
that some organic substances that pass a
0.45-p pore-size filter may be colloidal
rather than dissolved. Sharp (1973) used
a 0.025-p pore-size filter and an ultrafiltration membrane with a cutoff of 50,000 molecular weight (a nominal pore size of
about 0.003 ,x) to fractionate organic substances in the western North Atlantic
Ocean; he found that the 0.025-0.8-p size
fraction and the 0.003-0.025~ fraction each
contained about 8% of the total organic
material, and that about SO% of the total
passed the ultrafiltration
membrane. Ogura
( 1974) used ultrafiltration
membranes to
fractionate by molecular weight ( MW)
dissolved organic carbon (DOC) at two
stations in Tokyo Bay. There was a tendency for the [DOC] in the >lOO,OOO-MW
1 The research was supported
in part by National Science Foundation
grant DES72-01605
A02. Contribution
299 from the University
of
Georgia Marine Institute.
LIMNOLOGY
AND
OCEANOGRAPHY
fraction to be less than the [DOC] in the
<500-MW fraction in surface water; also
at least 40% of the DOC in all samples
was between 500 and 100,000 MW. I have
used ultrafiltration
equipment similar to
that of Ogura (1974) to fractionate by molecular weight organic substances in Georgia coastal water and have analyzed these
fractions chemically and spectroscopically.
The coast of Georgia is bounded by barrier islands. Between the mainland and
the islands are salt marshes and estuaries,
while on the seaward side of the islands is
continental shelf water extending off shore
about 100 km. Semidiurnal ebbing tides
flush water out of the marsh-estuarine system through the sounds between the islands and onto the continental shelf. After
some mixing on the inner shelf much of
the water that flushed out of the marshes
and estuaries returns on the incoming tide.
In contrast to the high [DOC] and intense yellow color of the marsh water, the
shelf water has a lower [DOC] and is a
less intense color.
Methods
Samples were collected at three marsh
stations, two sound stations, two nearshore
stations, and three offshore stations (Table
1) . Some stations sampled in the fall were
sampled again the following spring. All
samples were collected at slack high tide.
Marsh, sound, and 3-km-offshore stations
846
NOVEMBER
1976,
V.
21( 6)
Fractionation
Table
1.
Sample
G
stat ion
No.
1A
ik
5B
6~
ik
9F
10
11
12
13
14
15
I act
Ott
act
May
May
Ott
Ott
20 May
30 SCP
6 Ott
2.1 May
18 Fel,
18 Feb
20 May
21 day
3
15
16
5
7
G
F
H
1
I-I
J
and locations
N Lat
Date
2
20
Dates
74
74
74
75
75
74
74
75
74
74
75
75
75
75
75
jl'~3.8~
31'23.7'
31"23.6'
31°23.8'
31°23.71
31'23.2'
31'23.1'
31'23.2'
31'20.8~
31'21.4l
31'20.8~
31'30.0'
31'23.0'
31'30.0'
31°15.01
of
847
sampling.
Type OF
station
W Long
81'17.1'
81'17.2#
81°17.21
81°17.1'
81'17.2'
81'17.3'
81'17.3l
8lOl7.3'
81'14.5)
81'14.5'
81°14.51
80'57.0~
81°06.01
80'57.0'
81°03.01
by MW
3
3
3
15
15
15
23
Marsh
Marsh
Marsh
Marsh
Marsh
Sound
Sound
Sound
km offshore
km offshore
km oFFshore
km oFFshore
Ikm offshore
km offshore
km oFFshore
were sampled by immersing to a depth of
10 cm a precombusted (400°C) glass flask
sealed with a silicone stopper. Polyethylene gloves that were prewashed with soap
and water were worn. Care was taken to
avoid contamination from the small boat or
its engine by pointing the boat into the
wind and collecting water from the bow.
The stopper was released by hand, the
bottle was allowed to fill, and the stopper
was reinserted
before the bottle was
brought up through the surface to prevent
collection of surface film substances. It is
important to avoid collecting surface film
in any sampling whero concentrations of
subsurface particulate or dissolved organic
substances are wanted because substances
that accumulate at water-air
interfaces
tend to be different (frcqucntly higher molecular weight and may tend to easily coalescc into particulate material) from those
that remain in solution (Wheeler 1972a,b,
1975). Samples at the- 15- and 23-km-offshore stations were collected in 1.5-liter
PVC Niskin samplers at a depth of 1 m.
Samples from marsh, sound, and 3-km-offshore stations wero stored at 0°C and taken
immediately to the laboratory, where they
were
filtered
through
precombusted
(450°C) Rcevc Angel 984-H glass fiber
filters. The filtrates were then poured into
the ultrafiltration
cell, Samples from the
15- and 23-km-offshore stations were fil-
Fig. 1. Retention of organic substances by ultrafiltration
membranes UM-2 (A), PM-30 (B ),
and XM-10OA ( C ) . “/o Retention = { 1 - ( [DOC]
ultrafiltrate/[DOC]total
)} X 100.
tercd on board ship immediately after collection; the filtrates were kept at 0°C and
ultrafiltercd
in the laboratory
on land
within 4 h.
An Amicon model 402 (400-ml capacity)
ultrafiltration
cell was used with 76-mmdiameter Amicon ultrafiltration
membranes.
The membranes used were (with their
nominal
MW cutoffs in parentheses) :
WM-2 (1,000 MW), PM-30 (30,000 MW),
and XM-1OOA (100,000 MW). Thcsc membranes are organic polymers (polyelectroon porous,
lyte complexes ) supported
spongy polyethylene bases. They were calibrated with the following
organic compounds dissolved in doubly distilled water: urea (60)) dextrose ( ISO), sucrose
( 342)) raffinose ( 594)) inulin ( 5,000)) cytochromc c ( 12,384)) and bovine serum
albumen (65,000) (Fig. 1). The mcmbranes were, operated at pressures of 4.0
atm ( UM-2)) 0.5 atm (PM-30), and 1.0
atm (XM-IOOA).
These pressures yielded
ultrafiltrates at the rates lof about 1, 2, and
2 ml min-‘.
The ultrafiltration
mcmbrancs are reusable but must bc handled carefully bccause their surfaces can be easily scratched.
Before a membrane was used for the first
time it was placed in distilled water in a
4-liter beaker and stirred with a magnetic
stirring assembly for several hours, with
frequent changes of water. Then it was
Wheeler
848
Ultrafiltrate
volume (ml)
Fig. 2. Concentration
of organic substances in
the ultrafiltrate
is in part a function of degree of
concentration
of sample above membrane as ultrafiltration
proceeds.
placed in the ultrafiltration
cell, and 400
ml of distilled water was flushed through
it. This procedure had the effect of reducing the leaching of carbon from the
membrane into the ultrafiltrate
to a level
of <l mg C liter-l. The amount leached
during each experiment was determined,
and this amount was subtracted from the
ultrafiltrate
values of standards and samples. I found that the amount of organic
material that passed a membrane was a
function not only of molecular weight, but
also of the degree of concentration of the
sample above the membrane (Fig. 2).
Sample 9 was passed through the PM-30
membrane, and the ultrafiltrate
was collected in 50-ml increments and analyzed
for DOC. After 350 ml had passed the
membrane the final 50 ml was collected in
two &ml fractions. The results illustrate
the leakage of high MW substances
through the membrane on concentration of
substances above the membrane as ultrafiltration
proceeded. Leakage was found
with all three membranes (Ogura 1974),
but not until the volume of water above
the membrane was reduced by at least
50%. Therefore, each sample and standard
had an initial volume in the ultrafiltration
cell of 400 ml. The first 50 ml of ultrafiltrate was discarded, and the next 150 ml
was collected for analysis. The remaining
200 ml above the membrane in the cell
was discarded.
The percentage of each standard retained by each membrane was calculated
( Fig. 1). After filtration with a glass fiber
filter each sample was split into four aliquots : the first was ultrafiltered
with the
UM-2 membrane, the second with the PM30 membrane, and the third with the XM1OOA membrane. The fourth aliquot was
not ultrafiltered,
but was analyzed to provide total values for DOC, dissolved carand UV-visible
absorption.
bohydrate,
Concentrations of substances were calculated as by Ogura ( 1974).
I was concerned that any delay in ultrafiltration
might result in detectable microbial utilization of organic substances, so
I used the Reeve Angel 984-H filter, which
is highly efficient for removing small particles from marine samples (Sheldon 1972).
In a separate experiment, filtered water
from samples 1 and 9 was allowed to stand
at in situ temperatures for 8 h with no detectable decrease in concentrations of either total dissolved organic carbon or total
dissolved carbohydrate, indicating that the
4-5 h required to ultrafilter
the samples
did not allow detectable microbial decomposition. This does not mean that the organic substances investigated are refractory, but only that over this short period
the natural
filter-passing
heterotrophic
population did not use a detectable amount
of either total carbon or total carbohydrate.
Dissolved carbohydrate was determined
after Strickland and Parsons ( 1972)) dissolved organic carbon by the method of
Menzel and Vaccaro ( 1964). Visible and
ultraviolet
absorption spectra were obtained by placing each ultrafiltrate
in a
IO-cm spectrophotometer
cell and scanning from 700-240 nm against doubly distilled water as a reference with a Cary 14
recording spectrophotometer. Total absorption in the visible region of the spectrum
(to which the Gelbstoff or yellow color of
many coastal waters contributes)
was integrated with a planimeter into units of
square inches of spectrophotometer
chart
paper, and the percentage contribution of
Fractionation
400
I
500
Wavelength
(nm)
I
300
I
600
700
Fig. 3. Fractionation
of visible and ncar-ultraviolet absorption
spectrum of sample 7. Initial
glass fiber filtered
water sample (a); XM-100A
ultrafiltrate,
which contains substances <lOO,OOO
MW (b ) ; PM-30 ultrafiltrate,
which contains substances <30,000
MW (c);
and UM-2 ultrafiltrate, which
contains
substances
<l,OOO MW
(d).
each MW fraction to the total visible absorption (380-700 nm) of each sample was
calculated. Absorbance at 254 nm, a wavelength sometimes used to estimate concentrations of dissolved organic carbon in seawater (Mattson et al. 1974), was also noted.
Resdts and discussion
It can be seen in Fig. 1 that the membranes used do not retain 100% of substances with a MW above a precise cutoff
and 0% of substances below it. Molecular
shape is important in determining whether
a molecule will pass a given membrane, a
long narrow molecule having more tendency to pass than a spherical one of the
same MW (Amicon Corp. 1972). I-Iowever, inulin (a linear chain of about 30 p1:2-linked fructofuranosc units with a MW
of about 5,000) was retained 100% by the
UM-2 membrane, indicating that the reported results serve as a guide to molecular
weight despite retention differences due to
shape.
Figure 3 illustrates spectrophotometer
tracings from sample 7. Absorption in the
visible and near-UV regions illustrated is
by MW
849
due to organic substances ( Kalle 1966))
and thus the spectra illustrate the fractionation by MW of these in the sample. The
greatest absorption in both the visible and
UV regions was due to substances in the
l,OOO-30,000-MW fraction.
This fraction
was responsible for the greatest absorption
in all samples except sample 9, where the
>lOO,OOO-MW fraction predominated; sample 91also had a high [DOC] in the latter
fraction.
Figure 4 shows the percentage
contribution
of each fraction to the total
visible absorption of the filtered water. Despite large variations in total DOC and in
the intensity of color, the l,OOO-30,000-MW
fraction was responsible for at least 50%
of the total visible absorption in all except
sample 9. The <l,OOO-MW fraction consistcntly contributed less to the absorption
than the l,OOO-30,000-MW fraction.
The
abundance of yellow substances (Gelbstoff) in the >l,OOO-MW range was vividly illustrated during ultrafiltration
with
the UM-2 membrane of water from the
marsh, sound, and 3-km-offshore stations:
the ultrafiltrate
was colorless, while the
initially very pale yellow color of the water
above the membrane became more intense
as ultrafiltration
proceeded and substances
of higher molecular weight were concentratcd. Water above the UM-2 membrane
from the stations 15 and 23-km offshore
apparently remained colorless after concentration because color-forming
substances
were present initially
in very small
amounts. Figure 5 illustrates the absorbance at 254 nm for each MW fraction of
each sample. These absorbances have a
distribution
similar
to
[DOC],
but
[DOC]/Abs254
is not constant between
samples or within MW fractions of given
samples, indicating
differences in extinction at 254 nm for different organic substances.
Results of the chemical analyses are illustrated in Figs. 6 and 7. As shown by
the absorption data much of the organic
material at the marsh and sound stations
was in the l,OOO-30,000-MW fraction.
High concentrations of carbohydrate greater
than abiout 30,000 MW were found only in
850
Wheeler
I
MARSH
n
I5 and 23 km
’ OFFSHORE
1 SOUND
4
!i
f-
60
50 J
J&&...;
I 3 5 7 9 II I3 I5
MOLECULAR
WEIGHT
Fig. 4. Percentage contributions
of MW fractions
to visible absorption
due to filter-passing
substances. This absorption plus scattering
and absorption
by particulates
is responsible for color of the
water.
[Leftmost
group should read <l,OOO; rightmost
group should read >lOO,OOO.]
c
I
*
l.50-
:
l.25-
2
l.OO-
E
075-
3
0.500.25 -
l
il
n
NO. I 3 5 7 9 ll 13 15
1000
l 3
5 7 9 tl
IOOO- 30,000
MOLECULAR
Fig. 5. Absorbance
as in Fig. 4. [Leftmost
13 15 1 3 5 7 9’:
30,000-100,000
13 15 l
3 5 7 9 II
13 15
100,000
WEIGHT
at 254 nm by MW fractions of organic substances,
group should read <l,OOO; rightmost group should
lO-cm cell. Samples
read >lOO,OOO.]
same
Fractionation
851
by MW
5-
=3Ez
of
-
a
O2-
L
I
NO.
< 1000
IOOO-30,000
30,000-
Distribution
by MW
of dissolved
organic
the marsh. The marshes are dominated by
Spartina alterniflora Loisel., and the high
MW carbohydrates in the marsh may be
breakdown products of its cellulose cell
walls. Whatever their origin, high MW
> 100,000
WEIGHT
MOLECULAR
Fig. 6.
100,000
carbon.
Samples same as in Fig. 4.
carbohydrates may be removed from the
water (possibly through lysis into smaller
molecules by extracellular enzymes) rather
than transported out of the marsh; alternatively, much of the decrease in the con-
040
F
0.36
.c,
T
0.32
E
0.28
7
5
r
iif
0.24 -
4
020-
W
s
E
g
O.l60.12-
mo
5 0.08 u
8 88
--8 88
0.04-
L_.NO.
I
3
5
7 9 II I3 I5
< 1000
I 3 5
I 3 5 7 9 II I3 I5
IO00 - 30,000
MOLECULAR
Fig. 7.
Distribution
by MW
of dissolved
carbohydrate
30,000-100,000
I 3 5 7 9 II I3 15
> 100,000
WEIGHT
carbon.
Samples same as in Fig. 4.
852
Wheeler
centration of high MW carbohydrate between marsh and sound could be due to
dilution of these substances in the larger
volume of sound water.
Seaward from the marsh stations there
was a tendency for concentrations of DOC
and dissolved carbohydrate carbon (DCC )
to decrease. Much of the decrease in DOC
was in the 1,000-30,000-MW substances;
high concentrations
of these substances
were peculiar to the marsh water. The
offshore samples were variable with respect to which MW class contained the
most DOC. The concentration of DCC decreased in all MW classes, and at times
there was no detectable DCC in the
>30,000-MW
classes seaward from the
sounds.
This study illustrates the usefulness of
molecular filtration in studying the organic
chemistry of coastal water and in determining regional differences. Fractionation by
MW can be a useful tool for studying such
diverse phenomena as the chelation of
metals, and the utilization of organic matter as food by suspended heterotrophs, as
well as for concentrating high MW substances such as free enzymes from seawater.
References
AMICON CORP. 1972.
ington, Mass.
Applications
manual.
Lex-
KALLE, K. 1966. The problem of the Gelbstoff
in the sea. Oceanogr. Mar. Biol. Annu. Rev.
4: 91-104.
MATTSON, J. S., C. A. SMITH, T. T. JONES, S. M.
GERCHAKOV, AND B. D. EPSTEIN. 1974.
Continuous
monitoring
of dissolved organic
matter by UV-visible
photometry.
Limnol.
Oceanogr. 19: 536-535.
MENZEL, D. W., AND R. F. VACCARO. 1964.
The measurement
of dissolved organic and
particulate
carbon
in
seawater.
Limnol.
Oceanogr. 9 : 138-142.
OGURA, N. 1970. On the presence of 0.1-0.5
F dissolved organic matter in seawater.
Limnol. Oceanogr. 15: 476-479.
-.
1974. Molecular
weight
fractionation
of dissolved organic matter in coastal seawater by ultrafiltration.
Mar. Biol. 24: 305312.
SHARP, J. H. 1973. Size classes of organic carbon in seawater.
Limnol.
Oceanogr.
18:
441-447.
SHELDON, R. W.
1972. Size separation of marine seston by membrane and glass-fiber filters. Limnol.
Oceanogr. 17: 494-498.
STRICKLAND, J. D. H., AND T. R. PARSONS. 1972.
A practical
handbook
of seawater analysis,
2nd ed. Bull. Fish. Res. Bd. Can. 167.
WHEELER, J. R. 1972a.
Some effects of solar
levels of ultraviolet
radiation
on the dissolved organic constituents of surface waters.
Ph.D. thesis, Dalhousie Univ. 111 p.
-.
1972b.
Some effects of solar levels of
ultraviolet
radiation
on lipids
in artificial
seawater.
J. Geophys. Res. 77: 5302-5306.
-.
1975. Formation
and collapse of surface films.
Limnol.
Oceanogr.
20:
33S342.
Submitted:
Accepted:
8 #September 1975
27 April 1976