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.
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-|
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.
|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:
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:
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
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.
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).
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.
5 ml Dosimat titrator (Brinkmann)
calibrated iodine titration flasks
teflon-coated magnetic stir bars and retriever
squirt bottle with deionized distilled water (DDW)
magnetic stir plate
calibrated volumetric pipettes and flasks
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 12.41 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.
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.