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Tracer Monitored Titrations: Measurement of Dissolved Oxygen Todd 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) technique is evaluated for measurement of dissolved oxygen. The TMT developed in this work uses a simple apparatus consisting of a low-precision pump for titrant delivery and an optical detector based on a white LED and two photodiodes with interference filters. It is shown that the classic Winkler method can be made free of routine volumetric and gravimetric measurements by application of TMT theory, which allows tracking the amounts of titrant and sample using a chemical tracer. The measurement precision of the prototype setup was 0.3% RSD.

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INTRODUCTION Dissolved oxygen measurement is a cornerstone in natural water science. Due to its participation in several important biogeochemical reactions, the O2 molecule is often selected as the indicator used to examine the rate of primary production in the upper ocean,1 organic matter oxidation in the aphotic water column,2 and sediments,3 and it serves as a unique water mass identifier in ocean circulation and climate change studies.4,5 The standard method used to measure dissolved oxygen concentration, [O2], commonly known as the “Winkler Titration”, is an iodometric technique with a long history, stretching back well over a century.6 During this time, many significant developments have occurred, including, for example, optimization of the reagent concentrations and sampling techniques,7 and migration from a visual end point (using starch indicator) to end point detection based on both optical absorbance8 and redox state.9 Subsequently, fully automated systems were developed by a number of researchers.10,11 Modern developments outside of oceanography continue for many specialized municipal and industrial applications.12 A full review of sensor technology is beyond the scope of the present discussion, but it must be noted that oxygen sensors have a long history of development and use. In recent years, optical (luminescence lifetime-based) oxygen sensors13 have gained widespread acceptance as a viable alternative to the classic Clark electrode;14 both electrochemical and optical oxygen sensors are manufactured by many companies and used in a wide variety of applications. In light of the impressive advances in sensor technology, a common question arises: Why develop or improve antiquated titration methods? The central theme of our research development and use of autonomous chemical sensor technologieshas led to an appreciation that, as sensor use increases, the need for high-quality laboratory analyses does not diminish, because sensors must be calibrated and validated using trusted measurements. From our vantage point, the primary function of a sensor is not to eliminate standard © 2011 American Chemical Society

benchtop methods but to increase the number of measurements possible, effectively filling in the gaps between high quality 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 use of calibrated volumetric glassware and, preferably, automated titrant delivery and end point (ep) detection based on either an optical10 or amperometric16 sensor. The reactions involved in the iodometric determination of O2 are

Mn 2 + + 2OH− → Mn(OH)2

(1)

O2 + 4Mn(OH)2 + 2H2O → 4Mn(OH)3

(2)

2Mn(OH)3 + 2I− + 6H+ → 2Mn 2 + + I2 + 6H2O −

I2 + I ⇌

I3−

(3) (4)

I2 + 2S2O32 − → 2I− + S4 O6 2 −

(5)

IO3− + 8I− + 6H+ → 3I3− + 3H2O

(6)

The sample is collected by overflowing a vessel with the solution to be analyzed, adding MnCl2, NaOH, and NaI in excess, and immediately capping the vessel, typically with a ground glass stopper specific to the container. At this point, reactions 1 and 2 have quantitatively converted all O2 into an oxidized Mn precipitate and, upon addition of H2SO4, a stoichiometric amount of I− is oxidized to I2 (reaction 3). Due to the presence of excess I−, most of the I2 formed by reaction 3 Received: September 24, 2011 Accepted: November 29, 2011 Published: November 29, 2011 290

dx.doi.org/10.1021/ac202537f | Anal. Chem. 2012, 84, 290−296

Analytical Chemistry

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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). The optical ep detection method is based on observing the vanishing absorbance of I3−, which absorbs strongly at wavelengths below 500 nm (Figure 1).

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

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THEORY The TMT technique was originally developed to measure the total alkalinity of seawater,22 then later demonstrated in several other applications.21 In a TMT, a chemical tracer in the titrant or sample is used to calculate the amount of titrant added during each step of the titration. For the present work, Indigo Carmine (IC) was added to a thiosulfate solution and used to track the amount of titrant added. IC is a chemically inert substance, with no redox or pH transition occurring over the course of the titration. IC has a peak absorbance at 610 nm (Figure 1), where the absorbance of I3− is minimal but not negligible. As seen in Figure 1, spectral overlap must be considered at all wavelengths. Absorbance at wavelength λ is formally expressed as I−

I2 IC Aλ = ελ3 b[I− (7) 3 ] + ε λ b[I2] + ελ b[IC] where ε is the molar absorptivity and b is the optical path length. The absorbance due to the first two terms is difficult to separate, and in our case it is not necessary to do so. Because reaction 4 lies far to the right, we refer to the sum of both as the I3− 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

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

The iodometric titration thus described allows for accurate measurements of [O2] down to ∼50 μM, but errors due to atmospheric contamination during sampling and loss of I2 via volatilization during the titration become significant at lower [O2]. Fully automated modern titration techniques have been developed for low [O2] samples,18 but the most common technique used historically for measurement of dissolved oxygen in hypoxic (1%−30% saturation) and suboxic ( 2.0) but becomes linear as the ep is approached. IC contributes a small component to the measured A450, but since this channel is used only to locate the ep, relative IC contribution is clear in changes in A450 are sufficient. The A450 the post-ep absorbance data, which are nonzero with a positive 291



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In TMT, the abscissa is titrant dilution factor, f T, rather than volume. Replacing volume with f T in Figure 2 produces a plot identical in appearance, except that the x-data are dependent on absorbance measurements rather than buret volume. As the ordinate is also given in units of absorbance, the TMT results are wholly dependent on the performance of the optical system and not on the volumetric accuracy of titrant delivery or sample bottle. The dilution factor at the ep, f T(ep), is calculated from the slope and intercept of the two regression lines as

slope, consistent with the values predicted from the molar absorptivity of IC at 450 nm. The ep is calculated as the intersection of pre-ep and post-ep linear regressions of A450 vs titrant volume (conventional titration) or A450 vs dilution factor (TMT). We have found it convenient to define the intensity for the absorbance blank (I0) for A450 as the maximum intensity observed at 450 nm during the titration, resulting in data near the 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 I3− negligible at this wavelength (ε610 ∼ 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 the calculation of quantity of titrant added if no correction is made. A related complication results from definition of two different points in the titration for absorbance blank corresponding to the two optical channels. As discussed above, I0 for the 450 nm channel is defined as the maximum observed I0, which occurs very close to the ep. In contrast, the only logical choice of absorbance blank for the 610 nm channel is the untitrated I3− solution. However, A610 , relative to what would be a true blank solution with respect to both absorbers, is at a maximum in the untitrated solution. Several correction options are feasible. The most rigorous approach would combine accurate molar absorptivity data for I3− and IC with titration data, to iteratively I3− at each refine A610 by subtracting the contribution of A610 titration step. In the proof-of-concept presented here, this approach was not practical because titrations were carried out directly in nonuniform bottles with variable light paths. As a result of variability in sample container dimensions, we found it most suitable to apply a simple calibration based on a series of KIO3 standards (discussed below). The calculation approach involves estimating a dilution factor of the titrant, f T, from absorbance at 610 nm

fT (ep) =

[S2O3 ] A [IC] = ≈ IC 610 2 − [IC]0 [S2O3 ]0 ε610b[IC]0

(9)

Uncalibrated oxygen concentration is calculated as

f (ep)[S2O32 −]0 [O2 ]′TMT = T 4

(10)

where the factor of 4 represents the thiosulfate to oxygen stoichiometry (reactions 1, 2, 3, and 5). The calculations I3− . For the reasons outlined thus far do not account for A610 mentioned above, the system designed for this study is not amenable to direct absorbance corrections, even though the approximate magnitude of the absorbance interference is known and the effect is predictable. Instead, we elected to carry out this correction as a calibration of the analytical result in terms of [O2] and dilution factor. The full equation used to compute oxygen concentration following calibration is

[O2 ]TMT =

fT (ep)[S2O32 −]0 4

+ c0fT (ep)2

+ c1fT (ep) + c 2

(11)

⎛ [S O 2 −] ⎞ [O2 ]TMT = c0fT (ep)2 + ⎜⎜c1 + 2 3 0 ⎟⎟fT (ep) + c 2 4 ⎠ ⎝

(12)

or, equivalently,

2−

fT =

int pre − int post slope post − slope pre

(8)

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

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where the “0” subscript represents concentration in the titrant. IC In nonuniform vessels, as used here, the term ε610 b is the “effective molar absorptivity” and must be explicitly determined for each vessel due to differences between sample bottles and irreproducibility in bottle placement in the sample holder, which lead to an undefinable light path. This was accomplished in our study by adding an accurate pretitration volume of titrant to each sample bottle containing a known volume of sample. End point determinations in all TMT analyses discussed here are independent of volumetric measurements, with the caveat that the sample vessel was calibrated for effective molar absorptivity, pre-ep. Obviously, recalibrating every sample bottle in this fashion provides little advantage over a conventional titration. The purpose of the present work is to demonstrate that TMT theory is robust and can be applied to iodometric titrations. In this regard, our results are very encouraging and demonstrate that a TMT for dissolved oxygen is feasible and, if applied to a setup using a single titration vessel, offers a method free of routine gravimetric and volumetric measurements. It is noted that care must be taken to prevent I2 loss to volatilization when transferring the titrand from a sample bottle to a secondary titration vessel23 (see also Conculsions).

EXPERIMENTAL SECTION Chemicals. 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 from concentrated H2SO4 (Fisher Scientific, Certified ACS, 95−98% assay). The preparation guidelines for these three solutions are described in ref 24. The titrant was a solution of 8 mM S2O32− and 0.1 mM IC, prepared by dissolving Na2S2O3·5H2O (Fisher Scientific, Certified ACS 99.9% assay) in deionized water and then adding IC (Acros Organics, certified, assay not reported) from a 1 mM IC stock solution. Thiosulfate titrant was standardized, and the TMT system was calibrated using a stock solution of 0.00234 mol kg−1 KIO3, prepared from the salt (Fisher Scientific, Certified ACS, purity 99.5%; dried at 170 °C for 6 h) in deionized water (DIW). Titration results are reported on the molar scale (M = mol L−1). The densities used to convert between mass and volume were 0.9978 g mL−1 and 1.0233 g mL−1 for DIW and seawater, respectively. Apparatus. We first constructed a conventional titration setup, similar to many in use today, from an automated buret (Radiometer ABU901), a custom-built photometer consisting of a 473 nm LED (XLamp XPEBLU-L1-0000-00Y02-STAR, 292



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Cree), a 350 mA LED driver (RCD-24, Recom), a photodiode (PDB-C109, Advanced Photonix Inc.), and a transimpedance amplifier (MAX406) configured as a simple current-to-voltage converter. This system achieved routine precision for the measurement of dissolved oxygen on replicate seawater samples of 0.1% RSD. This setup was converted into a TMT system by replacing the blue LED with a high brightness warm white LED (BXRAW0240-00000, Bridgelux), adding a second photodiode, and placing interference filters over the photodiodes for wavelength selection. The output spectrum of the white LED is nonuniform because it is comprised of a blue LED with an organic phosphor coating, resulting in an intense peak at 450 nm and a broad band extending out to 800 nm. The interference filters (Intor Inc.) were chosen with center wavelengths (fwhm = 10 nm) corresponding to the 450 nm LED emission band and the 610 nm absorbance maximum of IC. The ABU901 autoburet was originally used in developing the TMT system, but it was later replaced by a high precision reciprocating pump (milliGAT LF, Global FIA). We found the milliGAT to be a convenient development tool because it can deliver precise volumes continuously, while the ABU901 is limited to 2 mL per stroke, upon which it must refill, leading to much lower sample throughput. The milliGAT delivery volume was calibrated using a gravimetric standard operating procedure.25 The milliGAT used in this study delivered volumes in the 1−5 mL range with precision consistently better than 0.3 μL with a repeatable error of −1.57 μL mL−1. The calibration function for the pump was based on 30 mass measurements of pure water delivered, 6 measurements each at delivery volumes of 1, 2, 3, 4, and 5 mL. The pump delivery volume was fit to the equation

vc = (0.99843)v − 2.8 × 10−4

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

Standards. A series of measurements using KIO3 are necessary to determine the concentration of S2O3 in the titrant in addition to a reagent blank.24 Solutions containing KIO3 were prepared by weighing an accurate amount (between 1 and 15 g, recorded to 0.0001 g) of the KIO3 stock solution into an accurately known amount of DIW. Conventionally, it is not necessary to quantity the amount of DIW during the standardization procedure, but in our case it was necessary in order to calibrate the bottle-specific TMT measurements. A single volume-calibrated glass-stoppered sample bottle was used for all standardization and blank measurements. Because it was not practical to dry the bottle in between each measurement, the most convenient approach to quantify the amount of DIW added to each standard was to first overflow and stopper the bottle filled with DIW (fixing the volume of DIW) and then remove ∼150 g of DIW by weight, just before the addition of KIO3. Next, a stir bar was added and reagents were added in the following order, with mixing in between each addition: 1 mL of H2SO4, 1 mL of NaOH/NaI, and 1 mL of MnCl2. Samples. Seawater was collected from the Scripps Pier in a 20 L carboy, sparged with room air for ∼2 h, and then stirred for ∼1 h. While stirring, samples were collected from the carboy by overflowing the sample bottle for ∼3 bottle volumes, followed by the addition of 1 mL each of the MnCl2 and NaOH/NaI reagents and then immediate stoppering of the sample bottle and shaking to mix the precipitate. Samples were stored overnight. After adding H2SO4, a stir bar was added and the bottle was placed on a stir plate, where it was mixed for ∼1 min. Similar to the procedure followed for the standard, a measured weight of the acidified sample (∼150 g) was removed from the bottle in order to provide a volume similar to the standard volume, where the ratio of S2O32−/IC in the titrant would generate absorbance values at both the 450 and 610 nm wavelengths within the linear range of the photometer. Titration Procedure. Following formation of I2 in the standard, blank, or sample, the bottle was placed into the bottle holder with the integrated photometer consisting of the LED and antipodal detector (Figure 3). The bottle holder sat on a stir plate which was operated at ∼400 rpm. PEEK tubing, connected to the pump, was placed into the sample bottle at a

(13)

where v and vc are uncalibrated and calibrated volume (in mL), respectively. On the basis of the linear fit, the standard error of the 30 pump calibration measurements was 0.29 μL (R2 = 0.996). Because small errors accumulate linearly in the reciprocating pump, the pump calibration equation is justifiably extended to the full volume range used in this study (1−20 mL). After completing the basic design using the milliGAT, a low-precision 50 μL solenoid pump (120SP1250, Bio-Chem Fluidics) was incorporated to deliver titrant in order to demonstrate that the TMT precision is not dependent on high accuracy or precision titrant delivery. A LabVIEW program was written to control the pumps and record voltage measurements from the light detectors. The ABU901 and milliGAT were controlled over a serial port, and the solenoid pump was operated from a DC relay that was triggered from the digital I/O of a NI USB-6210. Voltage was measured on a NI 9219. All titrations were carried out with a 10 s wait following the pretitration pulse and a 5 s wait following smaller pulses. The essential components of the system are shown in Figure 3. Glass bottles with ground glass stoppers (Wheaton, 300 mL, inner diameter ∼ 6.2 cm) were calibrated for volume contained24 and used for all standards and samples. Temperature corrections for volume contained and delivered were not necessary because all measurements were carried out within ∼1 °C of the temperature at the time of volumetric calibration. 293



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depth of ∼2 cm below the surface of the solution, taking care not to intersect the light path. Addition of titrant and collection of data were automated in LabVIEW, which saved a data file for each titration, consisting of a series of titrant volume increments with their corresponding photodiode voltages. Absorbance Spectra. I3− solutions were prepared by adding KIO3 standard to a solution of MnCl2, NaOH, NaI, and H2SO4 in seawater. IC measurements were made by adding indicator stock to acidified (H2SO4) seawater. Absorbance measurements were made in a 1 cm cuvette on an Agilent 8453 UV−vis spectrophotometer. Both sets of molar absorptivity values (Figure 1) were calculated as the slope of absorbance (1 nm resolution) vs concentration for a series of solutions.

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RESULTS AND DISCUSSION In 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 lower but still generally greater than 0.990. Fifteen iodate standards were titrated over a series of concentrations corresponding to the range of dissolved oxygen observed 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 during high latitude phytoplankton blooms). In this work we did not focus on suboxic concentrations characteristic of oxygen minimum zones. The iodate titrations were used to establish I3− a correction for the A610 interference by fitting a second order polynomial to a plot of [KIO3]std − [KIO3]TMT vs dilution factor (Figure 4). The rms error of the residuals was 0.45 μM, corresponding to 0.67 μM O2. The equation determined by the fit,

Δ[KIO3] = (1.598 × 10−3)fT (ep)2 − Figure 4. (A) Relationship used to calibrate the TMT system for errors due to spectral overlap at 610 nm. Δ[KIO3] is the difference between the known value of [KIO3] from the standard preparation and the value measured by the TMT with no 610 nm correction (eq 10). (B) Corrected and uncorrected results plotted vs standard [KIO3]; the line is 1:1.

−5 (1.666 × 10 )fT (ep)

(14) − 1.782 × 107 2 − (R = 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 more intuitive. However, absorbance corrections require accurate knowledge of path length and molar absorptivity (or effective molar absorptivity) and, as discussed above, in our system, these values are not easily separated. For example, molar absorptivity measured on a benchtop UV−vis is not directly transferable to systems with a broad bandpass (our system detects light at wavelengths ±10 nm from the transmission peak). Calibration of the instrumental response as a function of dilution factor is a robust approach that is independent of absolute absorbance, provided that the effective molar absorptivity of the system has been characterized. Following calibration, replicate samples were titrated on bottles collected over the course of ∼10 min from a carboy of seawater that was close to 100% saturation (Figure 5). The titration results indicate that the seawater was supersaturated by about 3% (relative to the temperature and salinity dependent O2 solubility26), which is not surprising, given that room air was rapidly bubbled into the seawater before sampling. Relative to the standard volumetric method, TMT measurements were high by 0.9 μM, or 0.4%. The RSD of the TMT measurements was also slightly higher than the standard method: 0.3%

compared to 0.1%, respectively. The first five TMT measurements were carried out using the high precision milliGAT pump while the last five were carried out with the low-precision solenoid pump. As expected, there was no observable degradation in precision with the shift to a lower precision pump and the mean calculated [O2] was not statistically different between measurements 1−5 and 6−10 (t test, P > 0.05). Although the 0.9 μM error in ∼100% saturated seawater is nearly within the 0.7 μM rms of the calibration curve, additional calibration runs and titrations alongside a standard benchtop system over a range of oxygen concentrations may be required to firmly establish TMT accuracy over the full range of [O2] in the ocean. Importantly, media effects on the dye will need to be taken into account if working over a range of salinity. In the example given here, the determination of effective molar absorptivity on a bottle-to-bottle basis accounted for the difference in extinction coefficient between the KIO3 standard and the seawater sample, but in a system where a number of samples are to be titrated over a range of ionic strengths using a single titrant standardization and dye calibration, the ionic strength dependence of the dye will come into play. 294



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

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CONCLUSIONS The overall level of performance obtained in this proof-ofconcept study is very encouraging. The TMT did not perform with quite the same level of precision as the conventional titration, but we suggest that the appropriate application of TMT to single-vessel measurements is the key to improving precision. The most important factor limiting TMT precision in this case was the bottle to bottle variability in effective molar absorptivity. In a single titration vessel with fixed optics, the optical path and bandpass are highly stable over the course of many measurements and effective molar absorptivity need only be determined infrequently. This was demonstrated previously over a 6 week period where hundreds of TMT measurements were carried out in a flow cell.22 Although the direct absorbance technique mentioned above is ostensibly simpler than any possible form of titration, the measurement requires a research-grade spectrophotometer (to access the narrow isosbestic wavelength of I2−I3−),17 resulting in a more expensive and more complex system than the TMT device we describe. Both the TMT and direct absorbance techniques will exhibit some sensitivity to temperature, due to temperature dependent molar absorptivity (and the I2−I3− equilibrium for the direct absorbance method). Previous work with other TMT methods indicates that such effects can be compensated once the temperature dependence of the molar absorptivity is established for the absorber and relevant equilibria.22 We see considerable merit in the direct absorbance method and believe that it may eventually supplant titrations as the preferred method for dissolved oxygen measurement. However, this has not occurred to date, probably due to the fact that, although spectrometers have been miniaturized,27 they remain somewhat costly and the portable versions do not provide the same levels of performance as benchtop instruments. Until cheap high-performance miniature spectrometers exist, direct narrowband measurements of the I2−I3− isosbestic point will require more expensive and more complicated hardware than required for high performance TMT measurements. Construction of a more practical titration system, though desirable, is not trivial and is beyond the scope of the innovation evaluated in this work. On the basis of our results, the primary improvement we recommend involves the use of a single titration vessel or, possibly, incorporating all aspects of the measurement (sampling, reagent addition, and titration) into a single vessel. Similar systems have been constructed by others, but we are not aware of a device that incorporates all of these steps into one container. For example, accurate trace level [O2] measurements typically require sampling and reagent addition to be carried out in the same vessel in order to achieve complete isolation from the atmosphere.18,19 The preferred configuration would be somewhat application-dependent. Analyzing a series of sample bottles would require transfer to a titration vessel. As mentioned above, transferal of the titrand must be done with care in order to avoid potential artifacts due to I2 loss to volatilization. As long as this issue is addressed, we expect that a TMT analyzer based on a single titration vessel would out-perform the system used in this study, because the bottle-to-bottle variability in effective molar absorptivity would be eliminated, lowering the overall uncertainty of the measurements.

Figure 5. Replicate titrations on seawater near atmospheric equilibrium (salinity = 33.5, T = 22.4 °C, [O2]100%sat = 219 μmol L−1). The line represents the average value of conventional titrations using a standard volumetric apparatus and gave [O2] = 226.7 ± 0.3 μmol 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) were carried out with a 50 μL solenoid pump (Biochem Fluidics).

Traditionally, a more concentrated thiosulfate titrant is used (e.g., 0.25 M vs 8 mM here). The primary advantage of the concentrated titrant is that it conveniently allows direct titration in the sample vessel without the need to remove any sample for displacement; the disadvantage is that titrant increments near the ep must be quite small (c.f. 1 μL). In our original volumetric-based setup, for example, the ep was typically around 1 mL while the glass stopper displaced ∼4 mL. In this demonstration, 50 μL titrant volume increments were made by both the high precision and low precision pumps, primarily because 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 high concentrations needed in order to add no more than ∼1 mL titrant while still obtaining reasonably high increments in A610. We did not systematically investigate IC solubility in variable concentrations of Na2S2O3, but such information would be useful 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 does not exhibit a particularly high molar absorptivity at 610 nm and it absorbs significantly at 450 nm (Figure 1), we have not yet found a more suitable inert dye. In addition to our work with IC, we also investigated the use of a redox indicator, methylene blue (MB), which transitions from colorless to blue at the ep (absorbance maximum ∼ 660 nm). It was hypothesized that use of a redox indicator in place of an inert dye could further simplify the technique by allowing use of a single wavelength photometer. Observation of the diminishing I3− absorbance would no longer be necessary, because the redox indicator’s absorbance will abruptly cut-on or cut-off at the ep. The basic idea was confirmed using a solution of Na2S2O3 + MB where absorbance at 660 nm was observed to cut-on at the ep and then increase linearly for subsequent additions of titrant. This modification to the TMT would require an alternative method of graphical analysis because there is no usable pre-ep data for 295



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(15) Emerson, S.; Stump, C.; Wilbur, D.; Quay, P. Mar. Chem. 1999, 64, 337−347. (16) Langdon, C. IOCCP Report No. 14; ICPO Publication Series No. 134; 2010. (17) Labasque, T.; Chaumery, C.; Aminot, A.; Kergoat, G. Mar. Chem. 2004, 88, 53−60. (18) Sahoo, P.; Ananthanarayanan, R.; Malathi, N.; Rajiniganth, M. P.; Murali, N.; Swaminathan, P. Anal. Chim. Acta 2010, 669, 17−24. (19) Broenkow, W. W.; Cline, J. D. Limnol. Oceanogr. 1969, 14, 450− 454. (20) Pai, S.-C.; Gong, G.-C.; Liu, K.-K. Mar. Chem. 1993, 41, 343− 351. (21) DeGrandpre, M. D.; Martz, T. R.; Hart, R. D.; Elison, D. M.; Zhang, A.; Bahnson, A. G. Anal. Chem. 2011, Article ASAP. (22) Martz, T. R.; Dickson, A. G.; DeGrandpre, M. D. Anal. Chem. 2006, 78, 1817−1826. (23) Culberson, C. H. WHP Operations Manual; WHPO: 1991. (24) Dickson, A. G. WHP Operations Manual; WHPO: 1996. (25) Dickson, A. G.; Sabine, C. L.; Christian, J. R. Guide to best practices for ocean CO2 measurements; PICES Special Publication 3; IOCCP Report No. 8; 2007. (26) Garcia, H. E.; Gordon, L. I. Limnol. Oceanogr. 1992, 37, 1307− 1312. (27) Smith, J. P. Anal. Chem. 2000, 72 (19), 653A−658A. (28) Peck, L. S.; Uglow, R. F. J. Exp. Mar. Biol. Ecol. 1990, 141, 53− 62.

The dissolved oxygen TMT introduced here would have broad applications in natural water research if the proof-ofconcept described above was modified into a more-convenientto-use setup. If a method was devised allowing easy transfer of the acidified sample into a single titration vessel, then the new method would offer operational simplifications to the analysis of the bottle samples collected using standard sampling procedures. Field analyses could be drastically simplified by the creation of compact portable systems that require no buret and use simple means of titrant delivery, such as plastic droppers or transfer pipets. In general, TMT approaches the accuracy and precision of conventional titration systems using simpler components. Once a sample cell and titrant solution have been standardized and characterized for effective molar absorptivity, the only requirement to obtaining high precision titration data is that the titrant can be delivered at sufficiently small, but not necessarily precise, steps. It is straightforward to scale the operational dilution factor to meet the requirement of different volumes of sample, buret, and pump by appropriately preparing 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 achieve high precision with the use of a micrometer syringe buret,28 but calibration and automation of the sample volume and microsyringe remain challenging. TMT can potentially scale down to very small volumes, leading to new micro-Winkler applications for emerging technologies such as lab-on-chip and Micro Total Analysis Systems.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected].

ACKNOWLEDGMENTS This work was supported by a stipend from the University of California Office of the President and by an REU supplement to NSF OCE 0844394.

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REFERENCES

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