DISSOLVED OXYGEN
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SUMMARY: Seawater is collected from known depths using
CTD-rosette sampling protocols. Subsamples are drawn
into precalibrated iodine flasks and dissolved oxygen is
chemically bound by the formation of a manganese (III)
hydroxide floc. The floc is subsequently dissolved under
acidic conditions which stochiometrically converts the
original dissolved oxygen (DO) oxidizing equivalents to
triiodide. The latter is quantitatively titrated with
sodium thiosulfate to a potentiometric end-point using
a high-precision computer-controlled titration system.
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1. Principle
The oxygen content of seawater is a fundamental measurement in
oceanography providing information which can elucidate water mass
movements, net primary productivity, atmosphere-ocean interactions
and carbon remineralization processes. The oxygen content of a
seawater sample is largely determined by a balance between: (a)
the exchange of atmospheric oxygen with the upper mixed layer, (b)
net increases due to photosynthetic processes and (c) net decreases
due to respiratory demands and heterotrophic processes. From an
oceanographic perspective, the measurement of dissolved oxygen is a
parameter of fundamental importance.
In this procedure, a divalent manganese solution and a strong
alkali are added to the water sample which results in the formation
of a floc (1). In the presence of oxygen the manganese II is
oxidized to manganese III (2). On subsequent acidification (1 < pH < 2.5),
the manganese hydroxide floc dissolves, and I2 is produced in
stoichiometric proportion to the original O2 concentration (3).
The iodine, in the form of triiodide (4), is titrated with a
standardized thiosulfate solution (5), and the oxygen content is
calculated from the quantity of thiosulfate consumed.
(1) Mn2+ + 2OH ---------- Mn(OH)2
(2) 2Mn(OH)2 + ½O2 + H2O ---------- 2Mn(OH)3
(3) 2Mn(OH)3 + 2I- + 6H+ ---------- 2Mn2+ + I2 + 6H2O
(4) I2 + I- ---------- I3-
(5) I3- + 2S2O32- ---------- 3I- + S4O62-
2. Precautions
It is extremely important to prevent contamination of the
sample with atmospheric oxygen during sampling, fixation and storage.
Drawing tubes and sample flasks must be free of bubbles (3.1 and
3.2.1), as must the autopipette system which dispenses the reagents
(3.2.2). If the water samples cool significantly or the seal dries
during storage, air can infiltrate the flask. The rims of the flasks
are filled with seawater and the samples are stored in a location
where temperature fluctuations are minimized (3.4).
The solubility of oxygen in seawater is temperature-dependent and
deep samples may warm up as they are brought to the surface (i.e.
through the thermocline). It is therefore necessary to measure the
temperature in the Niskin bottle at the time the sample is drawn in
order to calculate the oxygen concentration in situ (3.3). Oxygen
samples are always the first samples drawn from the Niskin bottles.
3. Field Sampling
3.1. Sample collection
3.1.1. Subsamples are drawn as soon as the rosette arrives on deck.
DO samples are the first samples drawn from the water
sampling bottle.
3.1.2. The drawing tube is flushed with sample and any bubbles are
displaced. The tube is then inserted (with water flowing)
almost to the bottom of the sample flask. Care is taken to
avoid creating turbulence in the flask in order to minimize
the intrusion of atmospheric oxygen.
3.1.3. The flask is overflowed with two to three volumes of sample.
With the sample flowing, the tube is slowly withdrawn from
the flask so that the bottle remains brimful when the tube
is completely withdrawn.
3.2. Sample fixation
3.2.1. Before fixing the sample, the iodine flask is carefully
examined for any bubbles that may have adhered to the walls.
If any bubbles are present, the sample is discarded and drawn
again.
3.2.2. Immediately after examining the flask, the two fixing
reagents (manganous chloride and alkaline iodide) are added
simultaneously using two 1 ml autopipettes mounted side-
by-side and activated by a single injection lever arm.
Care is taken so that bubbles are not added in this process.
The pipette tips are positioned at least 3 cm below the
surface of the sample, the lever arm is pressed slowly and
gently and released while the pipette tips are still
submerged, and the pipettes and tubing are checked frequently
for bubbles.
3.2.3. The glass stopper is carefully inserted and the flask is
shaken vigorously for at least 20 seconds with a rapid wrist
action (it is extremely important that the floc be dispersed
throughout the flask).
3.3. Sample temperature
3.3.1. The temperature of the water in the Niskin bottle is measured
and recorded immediately following sample collection and
fixation. This is accomplished by a second person to
reduce the time interval between sampling and temperature
determination.
3.3.2. An insulated holding container is rinsed thoroughly and
filled with the sample. The temperature is then measured
with a NBS traceable quick-response digital thermometer.
3.3.3. In situ temperature is measured at the time of bottle trip
using a calibrated thermistor (see Chapter 4).
3.4. Storage
3.4.1. Approximately 20 minutes after the samples are collected,
the sample bottles are shaken again to resuspend the floc.
The flared mouth of the flask is then filled with seawater
and bottles are stored in a cool location where temperature
fluctuations are minimized.
4. Analysis
4.1. Computer controlled potentiometric titration
The computer program (DO05HOT.BAS) is written in GW Basic and
located in the DO subdirectory on the AT 286 hard drive. This
program interfaces and operates the Dosimat (model 665) and pH
meter (Orion model 720A or EA940). The program parameters are
chosen to optimize the equivalence point determination based on
the computer controlled addition of µl quantities of titrant. The
titration time is approximately 10 minutes per sample, which is a
function of the response time of the electrode and the preset
electrode stability criteria. The titration time is minimized by
the manual addition of titrant by the operator until the color of
the sample is a very light yellow (almost clear). At this point
the computer-controlled functions are enabled. The computer
terminates the titration when the end point criteria are met.
The following steps outline the Incremental mv Titration program
operation:
1. After the computer has booted and all peripheral instruments
are connected to the computer and turned on, change
directories to C:\DO. The titration program and GWBasic
reside here. To initialize the program, type "GWBASIC DO05HOT"
and return.
2. If the system is ready to go (i.e., all connections are "OK"
and all peripherals are working) the program will list a
description of the task keys available. When you are done
with this screen, press return.
3. You will be presented with the option to load preset parameters
into the program. For work at Sta. ALOHA you should answer "Y"
or "y".
4. Next you will be asked to input the name of the file containing
the parameters. The default answers are ALOHA05 and C:\DO.
These default answers should be accepted for routine work at
Sta. ALOHA.
5. The program will list the default operational parameters which
have been found to optimize the sensitivity and precision of
the equivalence point determination at Sta. ALOHA and will
prompt you to either accept the default values or change them.
In most instances the statement is self-explanatory.
Statement Comments
Sodium thiosulfate normality (N) = 0.10 This is the standardized
normality of the Na2SO3
titrant. This together
with the change in mv and
volume added determines
the slope.
Buret size (ml) = 5 Capacity of the Dosimat
buret on the exchange
unit.
Titration increment size (ml) = 0.02 This is the initial
volume of titrant added
before the first slope
criteria is enabled.
Preadd volume (ml) = 0 This is set at 0 since
we add the preadd manually.
Total vol. of titrant (ml) = 4 This is one of the three
titration end point
criteria. If 4 ml of
titrant is reached, the
titration is terminated.
Stop potential (mv) = 100 This is the second of the
three end point criteria.
If the potential reaches
100 mv, the titration is
terminated.
Electrode drift time (sec/.2mv) = 5 Criteria for electrode
stability.
First slope increment change (mv/meq) = This is a calculated
value which controls the
reduction in titrant
increment size (300 mv/
titrant normality).
Second slope increment change (mv/meq) = calculated (750 mv/titrant
normality)
Third slope increment change (mv/meq) = calculated (3000 mv/titrant
normality)
First increment volume change (ml) = .01 Titrant volume increment
when first slope criteria
is enabled.
Second increment volume change (ml) = .002 Titrant volume increment
when second slope criteria
is enabled.
Third increment volume change (ml) = .0005 Titrant volume increment
when third slope criteria
is enabled.
6. If you wish to accept these values (which you should for routine
work at Sta. ALOHA), then enter a "Y" or "y" and return. If you
do not want to accept the default parameters enter a "N" or "n".
You will have to input all values except those calculated.
7. Next you will be asked to input:
(a) the date of analysis
(b) the pH meter model used
(c) if you have a printer connected to the computer
(d) if you want a detailed or summarized printout of the
titrations (use summarized for routine work at Sta. ALOHA).
(e) if you want an alarm to be activated at the end of each run
(f) the path of data storage (C:\DO\HOT##)
(g) the sample name. This is an 8 character max file name and
for standardization purposes the following convention is
used. For blanks and standards an alphanumeric designation
is used which represents the date and sample type. For
samples, an alphanumeric system is also used which
represents the iodine flask number and the HOT cruise
number (see examples below, assume the date of the analysis
is 3/25/92 for these examples):
Reagent Blank 3252bl1 (this creates a prn
file with the name 3252bl1.prn)
Primary KIO3 Standard 3252std1 (if you run more than
1 std then number sequentially)
CSK Certified KIO3 Standard 3252csk1
DO Sample 1H36 (iodine flask number
followed by HOT cruise number).
DO Sample Backtitrated 1BH36
Different DO Sample 1AH36
analyzed in a previously
used flask
(h) your name or ID
8. At this point a statement will appear on the screen instructing
you that the Dosimat is now in the manual titration mode and
to titrate your sample to a pale yellow color. To reduce the
titration time, manually titrate to a very pale yellow (almost
clear), then press "x".
9. From this point on the computer controls the titration,
adjusting the volume of titrant added in response to the
slope criteria. The titration is terminated when one of
three events occurs. Either the volume of titrant or mv
criteria is reached or the calculated slope increases four
consecutive times.
10. At this point, the results are printed out and you are
prompted to make any comments which are specific to that
sample. Record any problems, observations or concerns.
This is a great help in interpreting the data. Entering
return will close that file and you will be prompted if you
would like to run another sample. By entering "Y" or "y"
the process begins again. If you do not want to run any
more samples press "N" or "n" and you will see "ok". Type
"system" to return to the C: prompt. Type "run" to start
the program again. Normally the program is not terminated
until the end of the cruise. If all of the samples have
been run but more are to be collected, do not terminate
the program (i.e., do not enter "n"; leave it as is).
Other concerns:
(a) Backtitrating
The program adds incremental volumes of titrant to the sample
based on selected slope criteria. If the operator gets too close
to the equivalence point or passes it, during the manual titration
step, then the program will not reach its end point criteria and
will continue to run. To avoid this, check the mv reading. For
our samples this reading should be approximately 320-360 mv at the
end of the manual addition. If you find that you have titrated
just past this point, go ahead and enable computer controlled
titration and observe 3 or 4 cycles checking the slope. It should
decrease to a minimum value (~-500,000 to -900,000), and then
increase. If it initially increases and the mv readings are below
300 mv then you have gone too far. In this case, press F5 and add
volumetrically a known amount of primary standard. Ideally, you
want to add an amount which is equivalent to the normal manual
addition end point. I have found this to usually be around 100-
200 µl, depending on how far you went over or how long the program
ran. The program will have continued to add 20 µl if you passed
the end point during the manual addition. If you walk away and
come back 1/2 hr later, it will still be adding titrant at 20 µl
every 2-3 minutes. In this case the 200-300 µl of titrant have
been added. Therefore, you need to add enough primary standard
to equal this amount plus an amount equal to the manual addition
end point. Our primary standard is about 0.025 N and the titrant
about 0.05 N so you would need to add 400-600 µl of primary
standard to equal the added titrant, plus approximately 100 µl
to reach the manual addition end point. From this point, the
program should function properly.
(b) Interrupting an analysis in progress
If for some reason you need to interrupt an analysis, you can do
so by pressing F1.
(c) Restarting an interrupted analysis
After the buret refills, the program will prompt you whether you
want to run another sample. Continue in the normal manner.
(d) Volumetric delivery of standards
To obtain the maximum precision and accuracy it is critical to
deliver the primary standard in a manner which is accurate and
reproducible. At sea and in the laboratory we use class A
volumetric pipets which have been gravimetrically calibrated.
At the 5000 µl range, these glass pipets are accurate to within
+4.0 µl (0.08%) with a precision of +2.5 µl (0.05%). This is
equal to the precision of the Dosimat within the delivery range
of 500-3000 µl. To obtain this precision it is necessary to be
very reproducible in the delivery operation. A technique which
produces good results is outlined below:
1) Fill the pipet past the calibration mark and slowly deliver
the contents into the container until the meniscus is level
with the mark.
2) Invert the pipet and wipe off any excess solution from the
tip and barrel (the level of the solution within the pipet
will be below the tip at this point).
3) Invert again and touch the pipet tip to the sample vessel
wall. Release finger pressure and allow pipet to drain.
After the pipet has drained keep the tip against the vessel
wall for an additional 15-20 seconds.
4) Remove pipet and rinse any solution that may be on the vessel
wall into the sample with DDW.
To maintain accuracy and precision it is necessary to have an
absolutely clean pipet where none of the delivered solution clings
to the pipet walls. It is also good practice to rinse the pipet
with the filling solution prior to filling (unless this is the
first use of the pipet).
4.2. Reagent blank determination
The reagent blank is determined by adding the fixing reagents in
reverse order and titrating a known volume of primary standard
three separate times in the same flask. The volume of the first
titration includes the equivalents of primary standard added plus
any oxidizing or reducing substances in the reagents (V1); the
second titration should theoretically be equal to the equivalents
of primary standard only (V2). However, it has been our experience
that we rarely observe an obvious difference between V1 and V2
which could be attributed to the reagents and not a variable end
point estimation or pipetting. To help quantify this relationship
we have added a third aliquot titration (V3), which again should
be equivalent to only the primary standard. A statistical
comparison between the estimates of |V1-V2| and |V2-V3| is
performed to determine whether a significant blank exists.
Fill an iodine flask with distilled water and add in the following
order, mixing after each addition, 1 ml of sulfuric acid reagent
(10 N), 1 ml alkaline iodide reagent (4 M sodium iodide in 8 N
sodium hydroxide), and 1 ml manganese chloride reagent (3 M).
[Care must be taken to dispense the reagents below the blank
solution surface or in such a manner as to ensure that no
precipitate forms in the neck of the flask. If this occurs, a
high blank will be encountered if the precipitate subsequently
dissolves in the acidified blank solution during the titration
step. This is especially prevalent when using the redox
electrode which displaces a larger volume of sample into the neck
of the flask.] Add 1.0 ml of 0.01 N potassium iodate from a
precalibrated glass class A volumetric pipette. Titrate
manually, with adequate stirring, to a very pale yellow color,
enable computer operation of the titration and run to completion.
Record the volume at the equivalence point as V1. Add a second
1.0 ml aliquot of potassium iodate to the flask and titrate as
above.
Record as V2 the sum of the volume at the equivalence point and
the residual titrant from the first titration (this is equal to
the total amount of titrant added minus the volume of titrant
at the equivalence point). Add a third 1.0 ml aliquot of
potassium iodate, titrate and record volume as V3 in the same
manner as V2. Repeat this procedure a minimum of 3 times or
until a clear pattern is established.
4.3. Standardization of the thiosulfate titrant
Prepare a blank solution as outlined section 4.1. Add a known
volume (at least 5 ml) of potassium iodate primary standard (usually
0.02 N). It is necessary to know the volume added and the normality
of the potassium iodate to the highest degree of accuracy that is
reasonably possible. Therefore, class A volumetric pipets which
have been gravimetrically calibrated should be used and the potassium
iodate should be thoroughly dried, weighed in large aliquots relative
to the sensitivity of the balance to reduce weighing errors, and the
final primary standard volume determined gravimetrically. Titrate as
outlined above. Since precision is usually increased with increasing
number of titrations, it is our general practice to titrate 3-7
replicates at the beginning and end of each analytical run. In
addition to these, we typically run 1-4 standards per day since our
samples are analyzed at sea over a 3-4 day period.
4.4. Calculate the concentration of thiosulfate working solution
using the following relationship:
VT * NT = VI * NI
where: VT= the blank corrected volume of thiosulfate used to
reach the end-point
NT= the normality of thiosulfate solution
VI= the volume of potassium iodate added
NI= the normality of potassium iodate solution
4.5. Sample titration
Remove the ovelying seawater seal and remove the stopper. Add
1 ml of sulfuric acid reagent (10 N), and carefully slip a clean
teflon-coated magnetic stir bar into the flask. Place the flask
on a magnetic stirrer, insert the clean redox electrode and titrant
delivery tube. Titrate as outlined above. Record the presence of
bubbles or any unusual aspects of the titration or sample condition
in the comment section. Rinse electrode and titrant delivery tube
with DDW and repeat above procedure for the next sample.
5. Calculations
Calculate the DO concentration using the following formula:
(Vt-Vb) * Nt * E
µmol O2 l-1 = ________________ - RDO
(Vf - Vr)
where: Vt= volume of titrant (µl)
Vb= volume of blank (µl)
Nt= normality of titrant (µeq µl-1)
E= 0.2500 (µmol O2 µeq-1)
Vf= volume of flask (l)
RDO= dissolved oxygen content of the reagents = (0.804 * 0.14)/Vf
Vr= volume of fixing reagents (l)
This value is derived from Carpenter 1965 (i.e., DO content
of fixing reagents when 1 ml of each reagent is used in 140 ml of
sample equals 0.018 ml O2 l-1).
6. Precision and Accuracy
The method as outlined above is capable of a precision of 0.1%
or less (as defined by the coefficient of variation for triplicate
samples). The accuracy of the Winkler titration procedure, when
the Carpenter modifications are employed, has been determined to be
0.1% (Carpenter, 1965a,b).
Currently, we are using a high quality commercially prepared
potassium iodate certified reference standard to independently
assess the accuracy of our potassium iodate primary standard
normality. A batch of primary standard is prepared and the
theoretical normality normalized to the certified primary
standard value. Our primary standard is periodically checked
against the certified standard until the batch or lot is exhausted.
7. Equipment
5 ml Dosimat titrator (Brinkmann)
calibrated iodine titration flasks
teflon-coated magnetic stir bars and retriever
squirt bottle with deionized distilled water (DDW)
reagent dispensers
magnetic stir plate
calibrated volumetric pipettes and flasks
8. Reagents
Manganese chloride reagent (3 M)
Dissolve 600 g of manganese chloride tetrahydrate (MnCl2.4H2O)
in 800 ml distilled water and make up to 1 liter in a
volumetric flask.
Alkaline iodide reagent
Dissolve 320 g sodium hydroxide (NaOH) in 500 ml distilled
water and, separately, dissolve 600 g sodium iodide in 500
ml distilled water. Mix the he two solutions 1:1, by volume.
Sulfuric acid reagent (10 N)
Mix 280 ml of concentrated sulfuric acid into distilled water
using a 1 liter volumetric flask
Sodium thiosulfate reagent (0.05 N)
Dissolve 24.82 g of reagent grade sodium thiosulfate (Na2S2O3.
5H2O and make up to one liter with distilled water. Determine
exact normality as described under 4.3 above.
Potassium iodate reagent (0.025 N)
Dissolve 0.8918 g of dry (100°C, 2 hours) KIO3 into 800 ml
distilled water and bring up to 1 liter in a volumetric flask.
CSK 0.0100 N KIO3 1° standard
Wako Chemicals, Inc., 1600 Bellwood Rd., Richmond, VA 23237.
9. References
American Public Health Association. 1981. Standard Methods for
the Examination of Water and Wastewater, 15th edition.
Carpenter, J. H. 1965a. The accuracy of the Winkler method
for dissolved oxygen analysis. Limnology and Oceanography, 10,
135-141.
Carpenter, J. H. 1965b. The Chesapeake Bay Institute technique
for the Winkler dissolved oxygen method. Limnology and Oceanography,
10, 141-143.
CSK Certified Seawater Reference Standards. Sagami Chemical
Research Center, Sagimahara, Japan.
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