NOTES AND COMMENT
C
FIG. 3. Sampler in closed position:
A-pin;
B-closure
(plumbers’ force cup); C-rubber
drain
tube; D-metal
tab.
tact with PVC and rubber only. Finucane
and May (1961) replaced the force cup
closures on a Van Dorn water sampler
with rubber ball closures and reported
elimination of malfunctions owing to improper seating of the force cups. We have
experienced no malfunctions of this nature,
but different brands of force cups may seat
differently;
rubber ball closures may, in
general, improve our sampler.
In the cocked position, the closures are
held almost entirely out of the plane of
water sampled. The sampler is easily
flushed by swinging it laterally before
tripping.
The triggering mechanism is similar to
that used on most Ekman grabs and features large metal tabs that hook onto metal
THE WET
OXIDATION
OF
pins ( Figs. 2 and 3). The large tabs facilitate easy manipulation
even with heavy
gloves during winter sampling. The supporting line runs down through the center
of the triggering mechanism and is secured
by a knot between the triggering mechanism and the cylinder. The line may continue down to support several samplers to
be tripped in series. The auxiliary messenger can be supported by a nylon monofilament looped around the pin on the
triggering
mechanism which holds the
metal tabs.
In two years of intensive year-round
sampling, we have experienced virtually no
malfunctions with this sampler.
Mr. R. A. Scott constructed the sampler
and improved its design; Mr. R. Ristic
drew the figures.
RICHARD P. HOWMILLER
WILLIAM E. SLOEY
Center for Great Lakes Studies,
University of Wisconsin-Milwaukee,
Milwaukee
53201.
REFERENCES
FINUCANE, J. H., AND B. Z. MAY.
1961. Modified
Van Dorn
water
sampler.
Limnol.
Oceanog., 6: 85-87.
JOERIS, L. S. 1964. A horizontal
sampler for
collection of water samples near the bottom.
Limnol. Oceanog., 9 : 595-598.
SUMMERFELT, R. C., AND W. M. LEWIS. 1968.
A water sampler employing
a solenoid tripping mechanism.
Trans. Am. Fisheries Sot.,
97: 287-289.
VAN DORN, W. G. 1957. Large-volume
water
Trans. Am. Geophys. Union, 37:
sampler.
682-684.
WALKER, C. R.
1955.
A modification
Kemmerer water bottle for sampling
Progressive Fish Culturist,
waters.
of the
shallow
17: 41.
ORGANIC MATTER IN SEAWATER
Wilson ( 1961) introduced and Menzel
and Vaccaro ( 1964) subsequently refined
a comparatively
simple and long-awaited
method to measure the dissolved organic
carbon in seawater. It brings the attendant
problem of the completeness of the wet
oxidation the method incorporates. Prob-
ably not all organic compounds are fully
oxidized; however most biologists’ principal
interest lies in a restricted group of compounds : the biologically labile ones.
Within a restricted group of compounds
representative
members can be tested;
Menzel and Vaccaro did this by adding a
NOTES
AND
known amount of a compound to seawater
and measuring the increase in carbon dioxide. This technique has one possibly
serious defect. To detect a measurable increase in carbon dioxide one must add 2
to 5 mg/liter of the compound. This is
probably 100 to 1,000 times the natural
concentration of the compound in seawater.
If 99% oxidation were measured by this
method, the amount left unoxidized would
still be greater than the probable concentration of the compound in seawater. It is
difficult to predict whether the completeness of oxidation of a substrate will remain
constant when its concentration
is lowered two or three orders of magnitude,
especially in the case of the persulfate
oxidation where the kinetics are complicated by the rapid decomposition of the
persulfate during the reaction. Thus it is
not certain whether the results obtained in
this way are reliable.
This sort of approach has been commonly used in marine analytical chemistry
to test various oxidation methods (Fredericks and Hood 1965; Menzel and Corwin
1965; Armstrong and Tibbitts 1968); consequently it is important that this uncertainty be resolved.
The factors governing the extent of oxidation of organic material in the persulfate
wet oxidation have not been clarified and
it is not known whether the conditions
adopted by Menzel and Vaccaro are optimal. Different workers have used a variety
of temperatures for the oxidation ranging
from 1OOC (Wilson 1961) to 170C (Fredericks and Hood 1965), and from 100 to
400 mg of persulfate have been used by
different workers (cf. Menzel and Vaccaro
1964; Holm-Hansen et al. 1967).
A survey of the literature of persulfuric
acid oxidations revealed two relevant features of their chemistry. First, certain metal
ions, notably copper and silver, increase the
rate of oxidation of a variety of organic
compounds (House 1962; Ben-Zvi and Allen
1961). Presumably for this reason silver is
used as a catalyst for persulfate oxidation
of organic materials in freshwater analysis
( Leibnitz et al. 1962), although there ap-
293
COMMENT
pears to be no evidence that it actually
increases the extent of oxidation. The second and rather surprising feature was that
oxygen markedly inhibits the rate of oxidation of organic compounds by persulfuric
acid ( Ben-Zvi and Allen 1961).
I have developed a radiochemical procedure to study persulfate wet oxidation.
This method is comparatively simple and
has made it possible to study 1) the extent
of oxidation of biological compounds at
concentrations similar to those reported in
seawater, and 2) the effect of various
modifications of the procedure of Menzel
and Vaccaro on the extent of oxidation of
certain organic compounds.
METHODS
The radiochemical
method used was
briefly as follows: A known amount of 14Clabeled substrate was added to seawater
and then oxidized by the method of Menzel
and Vaccaro. After oxidation the carbon
dioxide was blo;wn off and the residual
radioactive material measured.
The oxidation procedure used was identical to that described by Menzel and
Vaccaro, with one exception: The gas used
to drive off the carbon dioxide before oxidation was oxygen, rather than nitrogen.
The sealed ampoule containing the oxidant,
seawater, and the radioactive substrate was
heated either at 130C (originally
in an
autoclave, but in later experiments in an
oil bath) or at 1OOCin a boiling-water bath
for 1 hr, unless specified otherwise. The
ampoule was then opened and the radioactive carbon dioxide removed by bubbling
a stream of carbon dioxide through the
combusted seawater. Then lOO- or 300-~1
samples of the combusted seawater were
placed on aluminum planchettes as a series
of droplets and evaporated to dryness at
1OOC; they were then timed for 1,000
counts with an end window
gas-flow
counter. For the fatty acids, the combusted solution after it was freed from
carbon dioxide was made alkaline before
spotting out, to prevent loss of material resulting from volatilization.
In these particular experiments stainless steel planchettes
294
TABLE
NOTES
1. Effect of substrate concentration
extent of its oxidation by persulfuric
acid
Added
substrate concn
( /.&liter
1
Glucose
Amino acid
mixture
Initial
radioactivity
( nCi/ml
)
AND
on
Residual
radioactivity
(%I
2,000
20’0
20
250
25
2.5
0.04
0.05
<0.025
4,000
400
40
10
10
10
3.8
3.7
3.8
were used. The residual radioactivity
is
reported as a percentage of the total origThe latter value was
inal radioactivity.
determined by spotting out uncombusted
samples, or, in later experiments, uncombusted samples containing an equivalent
amount of potassium sulfate in place of
persulfate (persulfate is converted to sulfate during the oxidation).
This procedure
is preferred because it avoids the probability that when the oxidizing agent is
added some of the organic material will be
oxidized during drying of the planchettes.
The seawater used in the work was taken
from station El in the English Channel
(50” 02’ N lat, 4” 22’ W long) and filtered
before use.
There is one defect to this method of
determining the effectiveness of the wet
oxidation procedure: Volatile organic combustion products will not be accounted for.
The amount of volatile organic products
was determined
by heating combusted
samples at 1OOC and bubbling nitrogen
through the sample. The gas was passed
through a condenser to remove water and
then through a trap cooled with solid carbon dioxide to remove most of the volatile
combustion products other than carbon dioxide. One ml of 1 N NaOH was added to
the trap and lOO+l samples dried and
counted. This procedure will determine
many, but not all, volatile combustion
products. With the amino acids, 0.1% or
less was detected as volatile products,
whereas with glucose no volatile products
were detected (0.01% was the limit of
detection).
It would thus appear that the
inability
to account for volatile organic
COMMENT
TABLE
2.
Extent
of oxidation
of substrates
persulfuric
acid
Added
concn
( bcz/liter
)
Substrate
Linolenic
acid*
Mannose
Aspartate
Glutamate
Glycine
Alanine
Valine
Leucine
Phenylalanine
Tyrosine
Serine
Threonine
Proline
Arginine
Histidine
* Combustion
lenic
acid where
40
1,300
27
24
30
23
19
17
15
16
28
24
18
23
21
temperature
was
it was 130C.
Residual
activity
by
radio(%)
4.5
<0.3,
0.8
4.0
0.5
0.5
1.4
6.4
2.6
0.6
1.3
0.6
22.0
4.4
1.2
lOOC,
except
for
lino-
combustion products is not a serious shortcoming.
RADIOACITVE
MATERIALS
All of the radioactive
materials were
obtained from the Radiochemical Centre,
Amersham.
None was further
purified
before use. The amino acid mixture (Ref.
No. CFB 104) used is reported to have the
following composition:
alanine lo%, arginine 6.5%, aspartic acid 9%, glutamic acid
12.5%, glycine 5%, leucine 12%, isoleucine
5%, lysine 5.5%, phenylalanine 7%, proline
6%, serine 5%, threonine 6%, tyrosine 3.5%,
and valine 7% (percentages are by radioactivity).
RESULTS
AND
DISCUSSION
Effect of substrate concentration
The oxidation of liC-labeled glucose and
an amino acid mixture by persulfuric acid
was determined at three substrate levels
(Table 1). Very little glucose remains
after oxidation. At the two higher glucose
concentrations
the residual radioactivity
was measurable and was less than 0.1%;
at the lowest concentration
no residual
radioactivity
could be detected, meaning
that the unoxidized glucose was less than
0.025%. Thus, apparently the glucose con-
NOTES
TABLE
oxidation
3.
Effect of various modifications
procedure on extent of oxidation
amino acid mixture
Residual
activity
Standard conditions*
Acid concn increased lo-fold
Saturated with Nz in place of 0,
lo-” CuSO added
Altered amounts of added persulfate:
30 mg
300 mg
AND
to the
of an
radio(%)
2.7-3.0
3.1
2.7
2.8
4.6
2.8-3.1
* These
were:
8 ml of filtered
seawater,
plus
40 pg/
liter
(10 nCi/ml)
of added
14C-labeled
amino
acids,
100
mg of K,S,Os,
200 pl of 3% H,PO,;
freed
from
CO, by
a stream
of oxygen,
sealed,
and heated
at 130C
for 1 hr;
opened
and radioactivity
determined
as described
in methods section.
centration is not dependent on the extent
of oxidation but is virtually
complete at
all concentrations tested. (It should be
noted that the radioactive glucose could
contain up to 2% impurities:
The Radiochemical Centre, Amersham, Data Sheet
9642).
The results obtained with the amino
acids are even more interesting. The extent
of oxidation is less, but remains constant
when the substrate concentration is varied
lOO-fold (Table 1). The agreement between these results is better than is usually
obtained between replicates.
These results imply that with the amino
acids and probably with glucose over the
range of concentrations studied, the extent
of oxidation is independent of substrate
concentration.
Thus, the results obtained
by Menzel and Vaccaro and also Fredericks
and Hood appear to be valid.
The results with a limited range of biochemical compounds (Table 2) also substantiate Menzel and Vaccaro’s findings
and, together with their results, imply that
biochemical compounds resulting from recent biological activity are extensively oxidized by acid persulfate,
under their
conditions.
Effect of alterations of the
combustion procedure
The measurement of soluble organic material in seawater by the method of Menzel
295
COMMENT
temperature
on
4. Effect of combustion
extent of oxidation of an amino acid mixture
TABLE
Temp
(“C)
80
100
110
130
Residual
activity
radio(%)
6.9
1.8
1.9
3.0
and Vaccaro is subject to comparatively
The source of this variahigh variation.
tion is uncertain, but it may lie in part
If this is the case,
with the oxidation.
a better understanding of the controlling
conditions of the wet oxidation would help
to reduce the overall variation
of the
method.
To this end the following alterations to
the oxidation procedure were examined :
1) either the persulfuric or phosphoric acid
concentration
was increased; 2 ) copper
ions were added; 3) the seawater was
freed from carbon dioxide before combustion with a stream of nitrogen in place of
oxygen; 4) temperatures other than 130C
were used. For these experiments the
amino acid mixture was used. It is easy
to work with, and more important, the
amino acids are incompletely oxidized so
that any improvements in oxidation conditions would be readily apparent. In all
these experiments, the total added amino
acid concentration was 40 pg/liter-of
the
order of their reported concentration
in
seawater (Degens, Reuter, and Shaw 1964;
Chau and Riley 1966).
From Tables 3 and 4 it is evident that,
with the exception of temperature, the
above alterations have little or no effect
on the extent of amino acid oxidation.
There was no evidence that added cupric
ions increased the extent of amino acid
oxidation;
it is possible, however, that
there are sufficient catalytic cations present in seawater already.
The amount of added persulfate does,
however, affect the amino acid oxidation.
With 30 mg of persulfate in place of the
usual 100 mg, more radioactivity remained
after oxidation.
There was perhaps less
residual radioactivity when 300 mg of per-
296
NOTES
AND
sulfate was used in place of 100, but the
results were somewhat variable in this
respect.
The temperature at which the wet oxidation was carried out, not surprisingly, had
a pronounced effect on the completeness
of the reaction. At the four temperatures
tested, most extensive oxidation occurred
at 1OOC rather than 130C (Table 4). This
observation was quite reproducible.
Substantially the same effect of temperature
was found when 300 mg of persulfate was
used in place of 100 mg and when linolenic acid was used in place of the amino
acid mixture.
The progress curve of the oxidation at
1OOC was determined; after 2.5 hr there
is little further breakdown of amino acids.
If the above findings with amino acids
and a fatty acid apply to biochemical
compounds in general, then the following
suggestions can be made of the best conditions for oxidation. The amount of added
phosphoric
acid used by Menzel and
Vaccaro is sufficient; nothing appears to
be gained by adding more. Nor is anything gained by adding copper ions as
catalyst. The oxidation proceeds to the
same extent when the reaction mixture is
saturated with either oxygen or nitrogen;
consequently either gas may be used to
flush the carbon dioxide from the seawater,
A suitable amount of oxidant is provided
by 100 mg of persulfate, for whereas with
300 mg a small increase in the extent of
oxidation may occur, this possible gain will
be offset by the resultant increase in blank
resulting from the extra added persulfate.
Of the temperatures studied, 1OOC appears
to be the most effective and it is in
many ways more convenient than 130C.
At lOOC, heating for 2.5 hr gives maximum
combustion.
The above conditions have been adopted
in our laboratory and are satisfactory, although it cannot be claimed that they have
caused any pronounced decrease in variation between replicates.
Intuitively
one
feels that this variation results from differences in heating, particularly the initial
rate of heating. This will probably be
COMMENT
greatest when the number of samples is
large and when the rate of heating is slow,
as it will be in an oven or autoclave. A
great advantage of 1OOC as the temperature of combustion is that a boiling-water
bath with a large heat capacity, ensuring
uniform heating of samples, can be used.
In conclusion, this work has established
that the customary method of determining
the effectiveness of an oxidation methodby measuring the extent of oxidation of
single substrates added at milligram quantities per liter-is
probably a reasonable one,
at least for the persulfuric acid method,
despite the fact that the substrates normally will be present only in microgram
quantities in seawater.
In addition, this work has confirmed the
findings of Menzel and Vaccaro that compounds such as sugars, amino acids, and a
fatty acid are on average oxidized to
more than 95% by persulfuric acid. With
the single exception of temperature, no
improvement could be made on the conditions originally
adopted by Menzel and
Vaccaro.
This does not help to resolve the uncertainty as to whether the wet oxidation
method oxidizes all the organic material
in seawater. Various workers disagree on
this fundamental point, and with the growing popularity
of Menzel and Vaccaro’s
method there is an urgent need to answer
this question.
P. J. LEB. WILLIAMSI
Department of Oceanography,
The University, Southampton,
England.
REFERENCES
ARMSTRONG, F. A. J,, AND S. TIBBITTS.
1968.
Photochemical
combustion
of organic matter
in sea-water,
for nitrogen,
phosphorus
and
carbon determination.
J. Marine Biol. Assoc.
U.K., 48: 143-152.
BEN-ZVI, E., AND T. L. ALLEN.
1961. The oxidation of oxalate ion by peroxidisulphate.
II.
The kinetics and mechanism of the catalysis
by Cu( 11).
J. Am. Chem. Sot., 83 : 43524357.
l I wish to acknowledge
the technical assistance
of M. W. Banoub and the financial
support of
the Natural Environment
Research Council.
NOTES
AND
&AU,
Y. K., AND J. P. RILEY.
1966. The deterDeepmination of amino-acids in sea-water.
Sea Res., 13: 1115-1124.
DEGENS, E. T., J. H. REUTER, AND K. N. F. SHAW.
1964.
Biochemical
compounds
in offshore
California
sediments and sea waters.
Geochim. Cosmochim. Acta, 28: 45-66.
FREDERICKS, A. D., AND D. W. HOOD. 1965.
A method for the determination
of dissolved
organic carbon in sea-water by gas chromatography.
Tech. Rept. 65-18T, Texas A & M
Res. Found., College Station, Texas.
HOLM-HANSEN,
O., J. COOMBS, B. E. VOLCANI,
AND P. M. WILLIAMS.
1967. Quantitative
microdetermination
of lipid carbon in microorganisms.
Anal. Biochem., 19 : 561-568.
HOUSE, D. A. 1962. Kinetics and mechanisms of
oxidations by persulphate.
Chem. Rev., 62:
185-203.
297
COMMENT
LEIBNITZ, E., U. BEHRENS, H. KOLL, AND H.
Zur chemischen
BestimRICHTER.
1962.
mung der gelosten organischen
Substanz in
Abwasser unter besonderer Berucksichtigung
der Peroxydisulfatmethode
bei der Analyse
Chem. Tech. ( Berlin),
von Schwelwasser.
14: 33-36.
MENZEL, D. W., AND N. CORWIN. 1965. The
measurement of total phosphorus in seawater
based on the liberation
of organically
bound
fractions
by persulfate
oxidation.
Limnol.
Oceanog., 10 : 280-282.
-,
AND R. F. VACCARO. 1964. The measurement of dissolved organic and particulate
carbon in seawater.
Limnol.
Oceanog., 9:
138-142.
of organic
WILSON,
R. F. 1961. Measurement
carbon in sea water.
Limnol. Oceanog., 6:
259-261.
THE DETERMINATION OF DISSOLVED ORGANIC CARBON IN SEAWATER:
A COMPARISON OF Two METHODS~
In connection with a study of the 13C :
12C ratios in the dissolved organic matter
in the sea ( Williams 1968)) there was an
opportunity to compare the wet oxidation
method of Menzel and Vaccaro ( 1964) for
determining
the total dissolved organic
carbon in seawater with the high energy
ultraviolet oxidation method of Armstrong,
Williams, and Strickland ( 1966).
There has been some uncertainty regarding the absolute values given by the Menzel
and Vaccaro method compared to that of
other workers. Skopintsev ( 1960 ) , Skopintsev and Timofeyeva ( 1962)) and Skopintsev, Timofeyeva, and Vershinina (1966)
report amounts of dissolved carbon several
times higher than those given by the
Menzel and Vaccaro method (Menzel 1964;
Holm-Hansen,
Strickland,
and Williams
1966; Barber 1967; Menzel and Ryther
1968; and unpublished data reports from
this institute ) .
Table 1 gives the results, comparing the
wet oxidation with persulfuric acid to the
ultraviolet oxidation on the same seawater
TABLE 1.
traviolet
’ This research was supported by U.S. Atomic
Energy Commission Contract No. AT ( 1 l-l ) GEN
10, P.A. 20.
Amazon
Depth
Wet oxidation
(A) compared with uloxidation (B) on the same samples
Organic
of
carbon
(mg
C/liter)
sample
(4
A
B
Surface
Surface
Surface
Surface
0.71
0.76
0.84
0.82
0.78
0.93
0.901
0.87
98
280
435
750
970
0.76
0.55
0.62
0.49
0.56
0.49
0.93
0.64
0.57
0.52
0.49
0.49
0.17
0.09
-0.05
0.03
-0.07
0.00
70
240
420
650
750
1,090
0.87
0.77
0.49
0.42
0.43
0.46
0.85
0.66
0.61
0.51
0.61
0.55
-0.02
-0.11
0.12
0.09
0.18
0.09
10
100
475
970
1,370
1,980
2,940
0.60
0.57
0.77
0.50
1.03
0.35
0.54
0.79
0.66
0.75
0.48
1.20
0.45
0.63
0.19
0.09
-0.02
-0.02
0.17
0.10
0.09
5
Mean
River
B-A
0.07
0.17
0.06
0.05
1.06
1.22
0.16
0.64
0.71
0.07