Tracer Monitored Titrations: Measurement of Dissolved OxygenTodd Martz,* Yuichiro Takeshita, Rebecca Rolph, and Philip Bresnahan

University of California, San Diego, Scripps Institution of Oceanography, La Jolla, California 92093, United States

ABSTRACT: The tracer monitored titration (TMT) techni-que is evaluated for measurement of dissolved oxygen. TheTMT developed in this work uses a simple apparatusconsisting of a low-precision pump for titrant delivery andan optical detector based on a white LED and twophotodiodes with interference filters. It is shown that theclassic Winkler method can be made free of routine volumetricand gravimetric measurements by application of TMT theory,which allows tracking the amounts of titrant and sample usinga chemical tracer. The measurement precision of the prototypesetup was 0.3% RSD.

■ INTRODUCTIONDissolved oxygen measurement is a cornerstone in naturalwater science. Due to its participation in several importantbiogeochemical reactions, the O2 molecule is often selected asthe indicator used to examine the rate of primary production inthe upper ocean,1 organic matter oxidation in the aphotic watercolumn,2 and sediments,3 and it serves as a unique water massidentifier in ocean circulation and climate change studies.4,5

The standard method used to measure dissolved oxygenconcentration, [O2], commonly known as the “WinklerTitration”, is an iodometric technique with a long history,stretching back well over a century.6 During this time, manysignificant developments have occurred, including, for example,optimization of the reagent concentrations and samplingtechniques,7 and migration from a visual end point (usingstarch indicator) to end point detection based on both opticalabsorbance8 and redox state.9 Subsequently, fully automatedsystems were developed by a number of researchers.10,11

Modern developments outside of oceanography continue formany specialized municipal and industrial applications.12 A fullreview of sensor technology is beyond the scope of the presentdiscussion, but it must be noted that oxygen sensors have a longhistory of development and use. In recent years, optical(luminescence lifetime-based) oxygen sensors13 have gainedwidespread acceptance as a viable alternative to the classicClark electrode;14 both electrochemical and optical oxygensensors are manufactured by many companies and used in awide variety of applications.In light of the impressive advances in sensor technology, a

common question arises: Why develop or improve antiquatedtitration methods? The central theme of our researchdevelopment and use of autonomous chemical sensortechnologieshas led to an appreciation that, as sensor useincreases, the need for high-quality laboratory analyses does notdiminish, because sensors must be calibrated and validatedusing trusted measurements. From our vantage point, theprimary function of a sensor is not to eliminate standard

benchtop methods but to increase the number of measure-ments possible, effectively filling in the gaps between highquality benchtop measurements.Using standard techniques, experienced analysts regularly

achieve [O2] measurement precision of 0.1% RSD or better,with similar accuracy.15 This level of performance requires useof calibrated volumetric glassware and, preferably, automatedtitrant delivery and end point (ep) detection based on either anoptical10 or amperometric16 sensor.The reactions involved in the iodometric determination of

O2 are

+ →+ −Mn 2OH Mn(OH)22 (1)

+ + →O 4Mn(OH) 2H O 4Mn(OH)2 2 2 3 (2)

+ +

→ + +

− +

+2Mn(OH) 2I 6H

2Mn I 6H O32

2 2 (3)

+ ⇌− −I I I2 3 (4)

+ → +− − −I 2S O 2I S O2 2 32

4 62

(5)

+ + → +− − + −IO 8I 6H 3I 3H O3 3 2 (6)

The sample is collected by overflowing a vessel with thesolution to be analyzed, adding MnCl2, NaOH, and NaI inexcess, and immediately capping the vessel, typically with aground glass stopper specific to the container. At this point,reactions 1 and 2 have quantitatively converted all O2 into anoxidized Mn precipitate and, upon addition of H2SO4, astoichiometric amount of I− is oxidized to I2 (reaction 3). Dueto the presence of excess I−, most of the I2 formed by reaction 3

Received: September 24, 2011Accepted: November 29, 2011Published: November 29, 2011

Article

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© 2011 American Chemical Society 290 dx.doi.org/10.1021/ac202537f | Anal. Chem. 2012, 84, 290−296

resides in the form of the triiodide ion, I3− (reaction 4).17

Thiosulfate is used to titrate the I2+I3− (reaction 5), and the

titrant is standardized with potassium iodate (reaction 6). Theoptical ep detection method is based on observing thevanishing absorbance of I3

−, which absorbs strongly atwavelengths below 500 nm (Figure 1).

The iodometric titration thus described allows for accuratemeasurements of [O2] down to ∼50 μM, but errors due toatmospheric contamination during sampling and loss of I2 viavolatilization during the titration become significant at lower[O2]. Fully automated modern titration techniques have beendeveloped for low [O2] samples,18 but the most commontechnique used historically for measurement of dissolvedoxygen in hypoxic (1%−30% saturation) and suboxic (<1%saturation) regions of the ocean has been one based on directabsorbance measurement of triiodide.19 Others have followedup on this work, demonstrating that direct absorbance can beused to measure oxygen over the full range of 0−100%saturation.17,20 The original development of this method wasdriven by the need to eliminate contamination of the sample bytrace levels of oxygen, while later developments were driven bythe desire for simplified measurement techniques (also themotivation for this work). Below, the Conclusions sectionbriefly contrasts tracer monitored titration (TMT) with thedirect absorbance method.One of the most critical aspects of achieving a high

performance oxygen titration system is meticulous calibrationof volumetric glassware, including a buret and set of groundglass stoppered sample bottles. Here we introduce aniodometric TMT for [O2] that requires no repeatedvolumetric/gravimetric measurements of sample or titrant. Anappealing aspect of this new technique is that it can beautomated using a cheap, low-precision pump to deliver titrantin place of the typical autoburet or, for more simplifiednonautomated measurements, titrant delivery can be mademanually without precision glassware (e.g., by plastic transferpipet).21 While volumetry and gravimetry will undoubtedlycontinue to serve as the criterion for the most accurate andprecise forms of titrimetry, there are a number of applicationswhere simplified titrations are needed. At sea, for example,where gravimetry is impossible and careful volumetric work iscomplicated by harsh conditions, a technique that can be made

to operate without the need for repetitive high precisionmeasurements of volume would be advantageous.

■ THEORYThe TMT technique was originally developed to measure thetotal alkalinity of seawater,22 then later demonstrated in severalother applications.21 In a TMT, a chemical tracer in the titrantor sample is used to calculate the amount of titrant addedduring each step of the titration. For the present work, IndigoCarmine (IC) was added to a thiosulfate solution and used totrack the amount of titrant added. IC is a chemically inertsubstance, with no redox or pH transition occurring over thecourse of the titration. IC has a peak absorbance at 610 nm(Figure 1), where the absorbance of I3

− is minimal but notnegligible. As seen in Figure 1, spectral overlap must beconsidered at all wavelengths. Absorbance at wavelength λ isformally expressed as

= ε + ε + ελ λ−

λ λ−

b b bA [I ] [I ] [IC]I3

I2

IC3 2 (7)

where ε is the molar absorptivity and b is the optical pathlength. The absorbance due to the first two terms is difficult toseparate, and in our case it is not necessary to do so. Becausereaction 4 lies far to the right, we refer to the sum of both as theI3− absorbance, but we recognize that a small amount of I2 is

likely to be present.In order to explain TMT theory, it is useful here to examine

sample titration data. Figure 2 presents typical TMT data for

air-saturated seawater using the system described below (seeMethods). A450 is initially outside the linear range of thephotometer (A > 2.0) but becomes linear as the ep isapproached. IC contributes a small component to the measuredA450, but since this channel is used only to locate the ep, relativechanges in A450 are sufficient. The A450

IC contribution is clear inthe post-ep absorbance data, which are nonzero with a positive

Figure 1. Molar absorptivity spectra for triiodide ion (I3−) and Indigo

Carmine (IC).

Figure 2. Typical data collected on a 150 mL seawater sample near100% O2 saturation. In order to illustrate the full range of absorbancevalues, 40 titrant additions at 0.5 mL were made. Operationally, it ismost expedient and improves ep detection to deliver one largepretitration pulse followed by smaller steps near the ep.

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slope, consistent with the values predicted from the molarabsorptivity of IC at 450 nm. The ep is calculated as theintersection of pre-ep and post-ep linear regressions of A450 vstitrant volume (conventional titration) or A450 vs dilution factor(TMT). We have found it convenient to define the intensity forthe absorbance blank (I0) for A450 as the maximum intensityobserved at 450 nm during the titration, resulting in data nearthe ep intersecting A = 0.A610 increases nearly-linearly with the addition of titrant

(Figure 2, R2 = 0.9985), but the absorbance of I3− is not

negligible at this wavelength (ε610I3−∼ 90 M−1 cm−1) and spectral

overlap leads to deviation from absorbance linearity at 610 nm,with respect to [IC], and produces significant errors in thecalculation of quantity of titrant added if no correction is made.A related complication results from definition of two differentpoints in the titration for absorbance blank corresponding tothe two optical channels. As discussed above, I0 for the 450 nmchannel is defined as the maximum observed I0, which occursvery close to the ep. In contrast, the only logical choice ofabsorbance blank for the 610 nm channel is the untitratedsolution. However, A610

I3−

, relative to what would be a true blanksolution with respect to both absorbers, is at a maximum in theuntitrated solution. Several correction options are feasible. Themost rigorous approach would combine accurate molarabsorptivity data for I3

− and IC with titration data, to iterativelyrefine A610 by subtracting the contribution of A610

I3−

at eachtitration step. In the proof-of-concept presented here, thisapproach was not practical because titrations were carried outdirectly in nonuniform bottles with variable light paths. As aresult of variability in sample container dimensions, we found itmost suitable to apply a simple calibration based on a series ofKIO3 standards (discussed below).The calculation approach involves estimating a dilution factor

of the titrant, f T, from absorbance at 610 nm

= = ≈ε

−

−fA

b[IC]

[IC][S O ]

[S O ] [IC]T0

2 32

2 32

0

610

610IC

0 (8)

where the “0” subscript represents concentration in the titrant.In nonuniform vessels, as used here, the term ε610

IC b is the“effective molar absorptivity” and must be explicitly determinedfor each vessel due to differences between sample bottles andirreproducibility in bottle placement in the sample holder,which lead to an undefinable light path. This was accomplishedin our study by adding an accurate pretitration volume of titrantto each sample bottle containing a known volume of sample.End point determinations in all TMT analyses discussed hereare independent of volumetric measurements, with the caveatthat the sample vessel was calibrated for effective molarabsorptivity, pre-ep. Obviously, recalibrating every samplebottle in this fashion provides little advantage over aconventional titration. The purpose of the present work is todemonstrate that TMT theory is robust and can be applied toiodometric titrations. In this regard, our results are veryencouraging and demonstrate that a TMT for dissolved oxygenis feasible and, if applied to a setup using a single titrationvessel, offers a method free of routine gravimetric andvolumetric measurements. It is noted that care must be takento prevent I2 loss to volatilization when transferring the titrandfrom a sample bottle to a secondary titration vessel23 (see alsoConculsions).

In TMT, the abscissa is titrant dilution factor, f T, rather thanvolume. Replacing volume with f T in Figure 2 produces a plotidentical in appearance, except that the x-data are dependent onabsorbance measurements rather than buret volume. As theordinate is also given in units of absorbance, the TMT resultsare wholly dependent on the performance of the optical systemand not on the volumetric accuracy of titrant delivery or samplebottle. The dilution factor at the ep, f T(ep), is calculated fromthe slope and intercept of the two regression lines as

=−−

f (ep)int int

slope slopeTpre post

post pre (9)

Uncalibrated oxygen concentration is calculated as

′ =−f

[O ](ep)[S O ]

42 TMTT 2 3

20

(10)

where the factor of 4 represents the thiosulfate to oxygenstoichiometry (reactions 1, 2, 3, and 5). The calculationsoutlined thus far do not account for A610

I3−

. For the reasonsmentioned above, the system designed for this study is notamenable to direct absorbance corrections, even though theapproximate magnitude of the absorbance interference isknown and the effect is predictable. Instead, we elected tocarry out this correction as a calibration of the analytical resultin terms of [O2] and dilution factor. The full equation used tocompute oxygen concentration following calibration is

= +

+ +

−fc f

c f c

[O ](ep)[S O ]

4(ep)

(ep)

2 TMTT 2 3

20

0 T2

1 T 2 (11)

or, equivalently,

= + + +−⎛

⎝⎜⎜

⎞⎠⎟⎟c f c f c[O ] (ep)

[S O ]4

(ep)2 TMT 0 T2

12 3

20

T 2(12)

where c0, c1, and c2 are empirically derived. Note that eq 12 iscomprised of constants and absorbance measurements only.

■ EXPERIMENTAL SECTIONChemicals. MnCl2 (3 M) and NaOH (8 M) + NaI (4 M)

were prepared from salts obtained from Fisher Scientific(Certified ACS, >99% assay). H2SO4 (5 M) was prepared fromconcentrated H2SO4 (Fisher Scientific, Certified ACS, 95−98%assay). The preparation guidelines for these three solutions aredescribed in ref 24. The titrant was a solution of 8 mM S2O3

2−

and 0.1 mM IC, prepared by dissolving Na2S2O3·5H2O (FisherScientific, Certified ACS 99.9% assay) in deionized water andthen adding IC (Acros Organics, certified, assay not reported)from a 1 mM IC stock solution. Thiosulfate titrant wasstandardized, and the TMT system was calibrated using a stocksolution of 0.00234 mol kg−1 KIO3, prepared from the salt(Fisher Scientific, Certified ACS, purity 99.5%; dried at 170 °Cfor 6 h) in deionized water (DIW). Titration results arereported on the molar scale (M = mol L−1). The densities usedto convert between mass and volume were 0.9978 g mL−1 and1.0233 g mL−1 for DIW and seawater, respectively.

Apparatus. We first constructed a conventional titrationsetup, similar to many in use today, from an automated buret(Radiometer ABU901), a custom-built photometer consistingof a 473 nm LED (XLamp XPEBLU-L1-0000-00Y02-STAR,

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Cree), a 350 mA LED driver (RCD-24, Recom), a photodiode(PDB-C109, Advanced Photonix Inc.), and a transimpedanceamplifier (MAX406) configured as a simple current-to-voltageconverter. This system achieved routine precision for themeasurement of dissolved oxygen on replicate seawater samplesof 0.1% RSD.This setup was converted into a TMT system by replacing

the blue LED with a high brightness warm white LED (BXRA-W0240-00000, Bridgelux), adding a second photodiode, andplacing interference filters over the photodiodes for wavelengthselection. The output spectrum of the white LED isnonuniform because it is comprised of a blue LED with anorganic phosphor coating, resulting in an intense peak at 450nm and a broad band extending out to 800 nm. Theinterference filters (Intor Inc.) were chosen with centerwavelengths (fwhm = 10 nm) corresponding to the 450 nmLED emission band and the 610 nm absorbance maximum ofIC.The ABU901 autoburet was originally used in developing the

TMT system, but it was later replaced by a high precisionreciprocating pump (milliGAT LF, Global FIA). We found themilliGAT to be a convenient development tool because it candeliver precise volumes continuously, while the ABU901 islimited to 2 mL per stroke, upon which it must refill, leading tomuch lower sample throughput. The milliGAT delivery volumewas calibrated using a gravimetric standard operatingprocedure.25 The milliGAT used in this study deliveredvolumes in the 1−5 mL range with precision consistentlybetter than 0.3 μL with a repeatable error of −1.57 μL mL−1.The calibration function for the pump was based on 30 massmeasurements of pure water delivered, 6 measurements each atdelivery volumes of 1, 2, 3, 4, and 5 mL. The pump deliveryvolume was fit to the equation

= − × −v v(0.99843) 2.8 10c4

(13)

where v and vc are uncalibrated and calibrated volume (in mL),respectively. On the basis of the linear fit, the standard error ofthe 30 pump calibration measurements was 0.29 μL (R2 =0.996). Because small errors accumulate linearly in thereciprocating pump, the pump calibration equation is justifiablyextended to the full volume range used in this study (1−20mL). After completing the basic design using the milliGAT, alow-precision 50 μL solenoid pump (120SP1250, Bio-ChemFluidics) was incorporated to deliver titrant in order todemonstrate that the TMT precision is not dependent on highaccuracy or precision titrant delivery.A LabVIEW program was written to control the pumps and

record voltage measurements from the light detectors. TheABU901 and milliGAT were controlled over a serial port, andthe solenoid pump was operated from a DC relay that wastriggered from the digital I/O of a NI USB-6210. Voltage wasmeasured on a NI 9219. All titrations were carried out with a 10s wait following the pretitration pulse and a 5 s wait followingsmaller pulses. The essential components of the system areshown in Figure 3.Glass bottles with ground glass stoppers (Wheaton, 300 mL,

inner diameter ∼ 6.2 cm) were calibrated for volumecontained24 and used for all standards and samples. Temper-ature corrections for volume contained and delivered were notnecessary because all measurements were carried out within ∼1°C of the temperature at the time of volumetric calibration.

Standards. A series of measurements using KIO3 arenecessary to determine the concentration of S2O3 in the titrantin addition to a reagent blank.24 Solutions containing KIO3were prepared by weighing an accurate amount (between 1 and15 g, recorded to 0.0001 g) of the KIO3 stock solution into anaccurately known amount of DIW. Conventionally, it is notnecessary to quantity the amount of DIW during thestandardization procedure, but in our case it was necessary inorder to calibrate the bottle-specific TMT measurements. Asingle volume-calibrated glass-stoppered sample bottle was usedfor all standardization and blank measurements. Because it wasnot practical to dry the bottle in between each measurement,the most convenient approach to quantify the amount of DIWadded to each standard was to first overflow and stopper thebottle filled with DIW (fixing the volume of DIW) and thenremove ∼150 g of DIW by weight, just before the addition ofKIO3. Next, a stir bar was added and reagents were added in thefollowing order, with mixing in between each addition: 1 mL ofH2SO4, 1 mL of NaOH/NaI, and 1 mL of MnCl2.

Samples. Seawater was collected from the Scripps Pier in a20 L carboy, sparged with room air for ∼2 h, and then stirredfor ∼1 h. While stirring, samples were collected from thecarboy by overflowing the sample bottle for ∼3 bottle volumes,followed by the addition of 1 mL each of the MnCl2 andNaOH/NaI reagents and then immediate stoppering of thesample bottle and shaking to mix the precipitate. Samples werestored overnight. After adding H2SO4, a stir bar was added andthe bottle was placed on a stir plate, where it was mixed for ∼1min. Similar to the procedure followed for the standard, ameasured weight of the acidified sample (∼150 g) was removedfrom the bottle in order to provide a volume similar to thestandard volume, where the ratio of S2O3

2−/IC in the titrantwould generate absorbance values at both the 450 and 610 nmwavelengths within the linear range of the photometer.

Titration Procedure. Following formation of I2 in thestandard, blank, or sample, the bottle was placed into the bottleholder with the integrated photometer consisting of the LEDand antipodal detector (Figure 3). The bottle holder sat on astir plate which was operated at ∼400 rpm. PEEK tubing,connected to the pump, was placed into the sample bottle at a

Figure 3. Schematic of the titration system. The sample bottle is fullyenclosed by the holder (drawn as a cutaway) and placed on a stir plate(not shown). In volumetric mode, only the milliGAT is operated. Todemonstrate TMT, the solenoid pump was added through a tee at thetitrant intake. See text for specific components associated with the I/Oand power relay.

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depth of ∼2 cm below the surface of the solution, taking carenot to intersect the light path. Addition of titrant and collectionof data were automated in LabVIEW, which saved a data file foreach titration, consisting of a series of titrant volumeincrements with their corresponding photodiode voltages.Absorbance Spectra. I3

− solutions were prepared byadding KIO3 standard to a solution of MnCl2, NaOH, NaI, andH2SO4 in seawater. IC measurements were made by addingindicator stock to acidified (H2SO4) seawater. Absorbancemeasurements were made in a 1 cm cuvette on an Agilent 8453UV−vis spectrophotometer. Both sets of molar absorptivityvalues (Figure 1) were calculated as the slope of absorbance (1nm resolution) vs concentration for a series of solutions.

■ RESULTS AND DISCUSSIONIn all analyses presented here, R2 values for pre-ep regressions(of A450 vs f T or v) were >0.999. Post-ep R2 values were lowerbut still generally greater than 0.990.Fifteen iodate standards were titrated over a series of

concentrations corresponding to the range of dissolved oxygenobserved in the ocean (13−220 μM KIO3 = 20−330 μM O2),with the upper limit chosen to roughly correspond with cold,fresh, supersaturated water (which commonly occurs duringhigh latitude phytoplankton blooms). In this work we did notfocus on suboxic concentrations characteristic of oxygenminimum zones. The iodate titrations were used to establisha correction for the A610

I3−

interference by fitting a second orderpolynomial to a plot of [KIO3]std − [KIO3]TMT vs dilutionfactor (Figure 4). The rms error of the residuals was 0.45 μM,corresponding to 0.67 μM O2. The equation determined by thefit,

Δ = × −

×

− ×

−

−

f

f

[KIO ] (1.598 10 ) (ep)

(1.666 10 ) (ep)

1.782 10

33

T2

5T

7 (14)

(R2 = 0.9997), was multiplied by 1.5 to convert from IO3− to

O2, and the resulting coefficients were used in eq 12, where c0 =2.397 × 10−3, c1 = −2.499 × 10−5, and c2 = −2.673 × 10−7.In the future, direct corrections to the absorbance measured

at 610 nm may be preferred, as this approach is somewhat moreintuitive. However, absorbance corrections require accurateknowledge of path length and molar absorptivity (or effectivemolar absorptivity) and, as discussed above, in our system,these values are not easily separated. For example, molarabsorptivity measured on a benchtop UV−vis is not directlytransferable to systems with a broad bandpass (our systemdetects light at wavelengths ±10 nm from the transmissionpeak). Calibration of the instrumental response as a function ofdilution factor is a robust approach that is independent ofabsolute absorbance, provided that the effective molarabsorptivity of the system has been characterized.Following calibration, replicate samples were titrated on

bottles collected over the course of ∼10 min from a carboy ofseawater that was close to 100% saturation (Figure 5). Thetitration results indicate that the seawater was supersaturated byabout 3% (relative to the temperature and salinity dependentO2 solubility

26), which is not surprising, given that room air wasrapidly bubbled into the seawater before sampling. Relative tothe standard volumetric method, TMT measurements werehigh by 0.9 μM, or 0.4%. The RSD of the TMT measurementswas also slightly higher than the standard method: 0.3%

compared to 0.1%, respectively. The first five TMT measure-ments were carried out using the high precision milliGATpump while the last five were carried out with the low-precisionsolenoid pump. As expected, there was no observabledegradation in precision with the shift to a lower precisionpump and the mean calculated [O2] was not statisticallydifferent between measurements 1−5 and 6−10 (t test, P >0.05). Although the 0.9 μM error in ∼100% saturated seawateris nearly within the 0.7 μM rms of the calibration curve,additional calibration runs and titrations alongside a standardbenchtop system over a range of oxygen concentrations may berequired to firmly establish TMT accuracy over the full range of[O2] in the ocean. Importantly, media effects on the dye willneed to be taken into account if working over a range ofsalinity. In the example given here, the determination ofeffective molar absorptivity on a bottle-to-bottle basisaccounted for the difference in extinction coefficient betweenthe KIO3 standard and the seawater sample, but in a systemwhere a number of samples are to be titrated over a range ofionic strengths using a single titrant standardization and dyecalibration, the ionic strength dependence of the dye will comeinto play.

Figure 4. (A) Relationship used to calibrate the TMT system forerrors due to spectral overlap at 610 nm. Δ[KIO3] is the differencebetween the known value of [KIO3] from the standard preparationand the value measured by the TMT with no 610 nm correction (eq10). (B) Corrected and uncorrected results plotted vs standard[KIO3]; the line is 1:1.

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Traditionally, a more concentrated thiosulfate titrant is used(e.g., 0.25 M vs 8 mM here). The primary advantage of theconcentrated titrant is that it conveniently allows direct titrationin the sample vessel without the need to remove any sample fordisplacement; the disadvantage is that titrant increments nearthe ep must be quite small (c.f. 1 μL). In our originalvolumetric-based setup, for example, the ep was typicallyaround 1 mL while the glass stopper displaced ∼4 mL. In thisdemonstration, 50 μL titrant volume increments were made byboth the high precision and low precision pumps, primarilybecause a 50 μL solenoid pump was readily available, hence the∼30-fold dilution of titrant. Although not a focus of this study,we observed a limitation of IC solubility at the highconcentrations needed in order to add no more than ∼1 mLtitrant while still obtaining reasonably high increments in A610.We did not systematically investigate IC solubility in variableconcentrations of Na2S2O3, but such information would beuseful for scaling iodometric TMTs in the future.If dye solubility is shown to be a limiting factor, then

alternative tracers may prove better than IC. Although IC doesnot exhibit a particularly high molar absorptivity at 610 nm andit absorbs significantly at 450 nm (Figure 1), we have not yetfound a more suitable inert dye. In addition to our work withIC, we also investigated the use of a redox indicator, methyleneblue (MB), which transitions from colorless to blue at the ep(absorbance maximum ∼ 660 nm). It was hypothesized thatuse of a redox indicator in place of an inert dye could furthersimplify the technique by allowing use of a single wavelengthphotometer. Observation of the diminishing I3

− absorbancewould no longer be necessary, because the redox indicator’sabsorbance will abruptly cut-on or cut-off at the ep. The basicidea was confirmed using a solution of Na2S2O3 + MB whereabsorbance at 660 nm was observed to cut-on at the ep andthen increase linearly for subsequent additions of titrant. Thismodification to the TMT would require an alternative methodof graphical analysis because there is no usable pre-ep data for

linear regression. We did not pursue this technique further butbelieve it is worth additional investigation.

■ CONCLUSIONSThe overall level of performance obtained in this proof-of-concept study is very encouraging. The TMT did not performwith quite the same level of precision as the conventionaltitration, but we suggest that the appropriate application ofTMT to single-vessel measurements is the key to improvingprecision. The most important factor limiting TMT precision inthis case was the bottle to bottle variability in effective molarabsorptivity. In a single titration vessel with fixed optics, theoptical path and bandpass are highly stable over the course ofmany measurements and effective molar absorptivity need onlybe determined infrequently. This was demonstrated previouslyover a 6 week period where hundreds of TMT measurementswere carried out in a flow cell.22

Although the direct absorbance technique mentioned aboveis ostensibly simpler than any possible form of titration, themeasurement requires a research-grade spectrophotometer (toaccess the narrow isosbestic wavelength of I2−I3−),

17 resultingin a more expensive and more complex system than the TMTdevice we describe. Both the TMT and direct absorbancetechniques will exhibit some sensitivity to temperature, due totemperature dependent molar absorptivity (and the I2−I3−equilibrium for the direct absorbance method). Previous workwith other TMT methods indicates that such effects can becompensated once the temperature dependence of the molarabsorptivity is established for the absorber and relevantequilibria.22 We see considerable merit in the direct absorbancemethod and believe that it may eventually supplant titrations asthe preferred method for dissolved oxygen measurement.However, this has not occurred to date, probably due to the factthat, although spectrometers have been miniaturized,27 theyremain somewhat costly and the portable versions do notprovide the same levels of performance as benchtop instru-ments. Until cheap high-performance miniature spectrometersexist, direct narrowband measurements of the I2−I3− isosbesticpoint will require more expensive and more complicatedhardware than required for high performance TMT measure-ments.Construction of a more practical titration system, though

desirable, is not trivial and is beyond the scope of theinnovation evaluated in this work. On the basis of our results,the primary improvement we recommend involves the use of asingle titration vessel or, possibly, incorporating all aspects ofthe measurement (sampling, reagent addition, and titration)into a single vessel. Similar systems have been constructed byothers, but we are not aware of a device that incorporates all ofthese steps into one container. For example, accurate trace level[O2] measurements typically require sampling and reagentaddition to be carried out in the same vessel in order to achievecomplete isolation from the atmosphere.18,19 The preferredconfiguration would be somewhat application-dependent.Analyzing a series of sample bottles would require transfer toa titration vessel. As mentioned above, transferal of the titrandmust be done with care in order to avoid potential artifacts dueto I2 loss to volatilization. As long as this issue is addressed, weexpect that a TMT analyzer based on a single titration vesselwould out-perform the system used in this study, because thebottle-to-bottle variability in effective molar absorptivity wouldbe eliminated, lowering the overall uncertainty of themeasurements.

Figure 5. Replicate titrations on seawater near atmosphericequilibrium (salinity = 33.5, T = 22.4 °C, [O2]100%sat = 219 μmolL−1). The line represents the average value of conventional titrationsusing a standard volumetric apparatus and gave [O2] = 226.7 ± 0.3μmol L−1 (n = 10). The calibrated TMT system measured [O2] =227.6 ± 0.7 μmol L−1 (n = 10). The first five TMT measurements(filled symbols) were carried out using a high precision pump(milliGAT), and the last five measurements (unfilled symbols) werecarried out with a 50 μL solenoid pump (Biochem Fluidics).

Analytical Chemistry Article

dx.doi.org/10.1021/ac202537f | Anal. Chem. 2012, 84, 290−296295

The dissolved oxygen TMT introduced here would havebroad applications in natural water research if the proof-of-concept described above was modified into a more-convenient-to-use setup. If a method was devised allowing easy transfer ofthe acidified sample into a single titration vessel, then the newmethod would offer operational simplifications to the analysisof the bottle samples collected using standard samplingprocedures. Field analyses could be drastically simplified bythe creation of compact portable systems that require no buretand use simple means of titrant delivery, such as plasticdroppers or transfer pipets. In general, TMT approaches theaccuracy and precision of conventional titration systems usingsimpler components. Once a sample cell and titrant solutionhave been standardized and characterized for effective molarabsorptivity, the only requirement to obtaining high precisiontitration data is that the titrant can be delivered at sufficientlysmall, but not necessarily precise, steps. It is straightforward toscale the operational dilution factor to meet the requirement ofdifferent volumes of sample, buret, and pump by appropriatelypreparing the titrant/tracer ratio.One particularly interesting application of TMT is for

titrations of very low volume samples (obtained from, e.g.,sediment pore waters). “Micro-Winkler” titrations can achievehigh precision with the use of a micrometer syringe buret,28 butcalibration and automation of the sample volume andmicrosyringe remain challenging. TMT can potentially scaledown to very small volumes, leading to new micro-Winklerapplications for emerging technologies such as lab-on-chip andMicro Total Analysis Systems.

■ AUTHOR INFORMATIONCorresponding Author*E-mail: trmartz@ucsd.edu.

■ ACKNOWLEDGMENTSThis work was supported by a stipend from the University ofCalifornia Office of the President and by an REU supplementto NSF OCE 0844394.

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Analytical Chemistry Article

dx.doi.org/10.1021/ac202537f | Anal. Chem. 2012, 84, 290−296296