2000_A sensor package for the simultaneous determination of nanomolar concentrations of nitrite, nit
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Ž.Marine Chemistry682000323–333www.elsevier.nl r locate r marchemA sensor package for the simultaneous determination ofnanomolar concentrations of nitrite,nitrate,and ammonia inseawater by fluorescence detectionRobert T.Masserini Jr.),Kent A.Fanning1UniÕersity of South Florida,Department of Marine Science,1407th AÕe.South,St.Petersburg,FL33701,USAReceived23February1999;accepted27August1999AbstractŽA fluorescence-based chemistry has been developed for the detection of nitrite and nitrate as excess nitrite following.reduction of nitrate to nitrite.Detection limits are4.6and6.9nM,respectively.The technique capitalizes on the triple bond between the two nitrogen atoms within the diazonium ion formed via the well-known reaction between an acidified nitrite sample and an aromatic primary amine.Fluorescence of p-electrons within this bond allows this reaction to be probed withŽ.standard fluorescence spectroscopy.Reverse Flow Injection Analysis rFIA is used to correct for background fluorescenceŽ.from leachates and naturally occurring dissolved organic matter parisons of samples analyzed for nitrite withwthis technique and with a highly-sensitive chemiluminescent method Braman,R.S.,Hendrix,S.A.,1989.Nanogram nitriteŽ.and nitrate determination in environmental and biological materials by vanadium III reduction with chemiluminescenceŽ.xŽdetection.Analytical Chemistry,61242716–2718showed excellent agreement between the two methods slope s0.9996 2.and r s0.9956.These fluorescent nitrite and nitrate q nitrite chemistries were coupled in a sensor package with awmodified version of a fluorescent ammonia chemistry Jones,R.D.,1991.An improved fluorescence method for theŽ.x determination of nanomolar concentrations of ammonia in natural waters,Limnology and Oceanography.364814–819, which also has a nanomolar detection limit.The throughput rate of the fully automated three-channel instrumentation is18 samples per hour.A field experiment demonstrated the capability of the nutrient sensor package to determine horizontal gradients in nitrate,nitrite,and ammonia in oligotrophic surface waters.q2000Elsevier Science B.V.All rights reserved.Keywords:nutrients;nitrate;nitrite;ammonia;fluorescenceCorresponding author.Fax:q1-727-553-1189;E-mail: masserin@1E-mail:kaf@.1.Introduction1.1.BackgroundNitrogenous solutes such as ammonia,nitrite,and nitrate are thought to play a major role in limiting phytoplankton primary production in oceanic surface0304-4203r00r$-see front matter q2000Elsevier Science B.V.All rights reserved.Ž.PII:S0304-42039900088-2()R.T.Masserini Jr.,K.A.Fanning r Marine Chemistry682000323–333 324Žwaters over short time scales Ryther and Dunstan, 1971;Garside,1985;and Jackson and Williams, .1985.The extremely low concentrations of these solutes in the upper10m of most of the central oceanic gyres are consistent with this conclusion and constitute a principal reason that these waters are considered oligotrophic.Because food webs in these regions are critical to the oceanic ecosystem, the concentrations of nitrogenous nutrients in oligotrophic waters,the mechanisms by which they are utilized and transformed,and their flux rates between oceanic reservoirs are nonetheless of con-Žsiderable ecological interest Codispoti and Chris-.tensen,1985.Analytical methods for the determination of the various forms of inorganic nitrogen-bearing nutrients in oligotrophic waters should be highly sensitive Ž.less than10-nanomolar detection limit,automated, and have a high sample throughput rate.The re-search described here was part of an effort to de-velop reasonably simple automated chemical tech-niques that meet these criteria and are ultimately adaptable for the in situ determination of nitrate, nitrate,and ammonia in oligotrophic seas by a au-Ž.tonomous underwater vehicle AUV.The standard colorimetric methods commonly used in the determination of nitrate concentrations in Žseawater e.g.,Bendschneider and Robinson,1952; Grasshoff,1976;ALPKEM,1988;and Gordon et al., .1993are limited to a realistic detection limit of approximately200nanomolar,which is frequently higher than the concentration of nitrate within theŽ.oceanic euphotic zone Fanning,1989.Most of the published high-sensitivity methods for the determina-Žtion of nitrate and r or nitrite in seawater e.g.,Axel-rod and Engel,1975;Garside,1982;Motomizu et al.,1986;Oudet and Montel,1988;Braman and Hendrix,1989;Kieber and Seaton,1995,Yao et al., .1998were found not to be readily adaptable for in situ application or to be too cost-prohibitive,com-plex,or limited in dynamic range.Based on the success of the fluorescent,high-Ž.Ž.sensitivity;1nM Jones1991method for am-monia in seawater,a fluorescence-based approach was selected because it avoided the above difficul-ties.In addition,the in situ requirement also meant that flow-injection techniques had to be adopted because the gas-segmented stream flow of many of the standard automated colorimetric chemistries would likely be rendered unacceptably erratic by in situ hydrostatic-pressure effects on the bubbles.This report describes the initial stage of the re-search:a laboratory r shipboard version of a nutrient sensor package that can determine oligotrophic con-centrations of nitrate,nitrite,and ammonia simulta-neously,either in a series of individual seawater samples or by repetitive sampling of a continuous stream of surface seawater.We first present the theory and operational parameters for fluorescent methods for nitrate and nitrite,then a brief descrip-Ž.tion of modified version of the Jones1991ammo-nia method in the sensor package,and finally an example of the type of field data that the package can deliver.2.Fluorescent analysis of nitrite and nitrate2.1.TheoryŽ.The basis for the nitrite and nitrate analyses is a well-known reaction in synthetic organic chemistry, namely acidification of nitrite to form the nitrosium ion and its subsequent combination with a primaryŽaromatic amine to form a diazonium ion Seyhan, .1989.The aromatic portion of the diazonium ion is readily probed with standard fluorescence spec-troscopy because of the conjugated double-bond structure that contains p-bonds.Electrons within this type of bond,p-electrons,readily fluoresce.In addi-tion,the diazonium ion contains two p-bonds in the triple bond between the two nitrogen atoms;there-fore the change in the molecular structure resulting from the formation of the diazonium ion will en-hance the fluorescence response above that provided by the amine alone.The primary aromatic amine selected for this method was the simplest one:aniline.Thus,the fluorescent response that makes the method possible comes as a result of the formation of the benzenedia-zonium ion.The magnitude of the response is a function of the nitrite concentration present.As in established nitrate techniques,nitrate is measured as excess nitrite formed when nitrate is quantitatively reduced to nitrite in a copper-activated cadmium reduction column.Reduction efficiency depends upon()R.T.Masserini Jr.,K.A.Fanning r Marine Chemistry682000323–333325the flow rate through the column,the bed volume ofŽthe column,and the pH of the solution Nydahl, .1976.The wavelength of fluorescent excitation for a molecule is related to its absorbance spectrum.Some of the ultraviolet and visible wavelengths at which a given molecule exhibits absorbance maxima are the wavelengths that promote electrons within a conju-gated double bond structure to the excited p U state within the system.Absorbance peaks to be consid-ered are those that formed because of the molecular transformation in the reaction of analyte with reagent. The wavelength of each absorbance peak is used in succession as the excitation wavelength,and the wavelength of maximum emission associated with it is determined in the same fashion as the wavelength of maximum absorbance in absorbance spectroscopy.2.2.Optimization of method parameters2.2.1.WaÕelengthsOptimal wavelengths for detection of the nitrite-derived benzenediazoniuim ion were determined byŽ.first measuring the excitation or absorbance spec-trum of the reaction between nitrite and aniline.The aniline reagent solution selected for this experiment was500m l of aniline dissolved in1l of10%HCl Ž.volume,and a background excitation r absorbanceŽ. spectrum was obtained from a1:1V:V mixture of this aniline reagent solution and deionized water Ž.DIW.Next,the excitation r absorbance spectrum for a benzenediazonium sample solution was mea-Ž.sured for a1:1V:V mixture of the aniline reagent solution with a1000nM solution of nitrite in DIW. The difference between these two spectra yielded the absorbance r excitation spectrum of the reaction. Wavelengths of the maxima in the absorbance r exci-tation spectrum of the reaction were used as excita-tion wavelengths,and the emission spectra of the two mixtures described above were obtained for each excitation wavelength.Subtraction of the emissionŽ.spectrum of the first or background solution from that of second solution yielded the emission spec-trum for the reaction.From these spectra it was determined that the excitation wavelength of220nmŽ. produced the largest emission peak295nm for the reaction.2.2.2.TemperatureŽDue to the short period of time approximately40 .s that the nitrite–aniline reaction mixture is within the analytical manifold,heating is required to in-crease the yield.Since the solubility of a gas de-creases as temperature increases,DIW wash and HCl reagents were sparged with helium for30min throughŽ. polytetrafluoroethylene PTFE tubing to replace as many of the dissolved atmospheric gases present as possible with less-soluble helium and thus reduce the likelihood of bubble formation during heating.Opti-mum temperature setting of the heat exchanger was determined to be708C,which yielded an effluent temperature of508C.Higher temperature settings gave greater reaction yields but also increased the noise within the analytical system.The708C setting was a compromise that provided sufficient sensitivity Ž.less than10nM nitrite and nitrate with a reason-able noise level.Lengths of microporous PTFE tub-ing were placed just downstream of the heat ex-changers to permit the escape of any bubbles that form because of temperature-induced decreases in Ž.solubility Karlber and Pacey,1989.2.2.3.Manifold lengthThe length,internal diameter,and coiling of the manifold tubing,as well the flow rates within the tubing,control the dispersion of sample and reagent after the flow injection of sample into the carrier reagent stream has ually,a greater length means a higher degree of reaction completion be-cause of increased reaction time.However,a longer manifold also means more time required per sample and thus a lower sample throughput rate.It also produces a greater dilution of the injected solution by the carrier stream and thus a lower sensitivity due to increased dispersion.FIA methods are therefore devised to minimize the extent of dispersion of the sample into the carrier stream,and hence minimize the dilution of the sample by the carrier stream. Usually,FIA methods restrict manifold length to1m Ž.or less Ruzicka and Hansen,1988.However,tests described below demonstrated that Ž.nitrite and nitrate determinations in seawater are more accurately performed by reverse-flow injection Ž.analysis rFIA,and,in rFIA,dispersion should be maximized.The carrier is the sample and the greater()R.T.Masserini Jr.,K.A.Fanning r Marine Chemistry682000323–333 326the dispersion of the reagent that is injected into the carrier r sample the greater the extent of the reactionŽ. and the resulting signal Johnson and Petty,1982. After many experiments,the competing criteria forŽ.the optimization of reaction extent sensitivity and sample processing rate indicated that the total tubingŽ. length for the nitrite and nitrate manifolds Fig.1Ž.should be approximately5m0.8mm i.d..2.2.4.Interferences and reagent contribution to fluo-rescenceAs mentioned,our fluorescent nitrite r nitrate tech-nique was initially developed as a typical FIA method involving the injection of a seawater sample into a carrier stream and the subsequent addition of reagents.An at-sea intercomparison of this FIA method and the chemiluminescence method of Bra-Ž.man and Hendrix1989demonstrated a significant Ž.offset on the order of100’s nM difference between the nutrient concentrations reported by the two tech-niques.FIA results were always higher.Interest-ingly,the offset decreased over time,and this tempo-ral decrease in the offset between the two techniques was found to be correlated with the age of Tygon pump tubing used to propel solutions through the boratory investigations found that dis-Ž.solved organic matter DOM from a variety of sources in seawater fluoresces at the same wave-Žlengths as the benzenediazoniuim ion220-nm exci-.tation and295-nm emission.Thus,some of the fluorescent signal in the seawater stream came from organic matter in the seawater plus that released from Tygon tubing.To correct for this false DOM signal,rFIA was implemented.As mentioned,in rFIA the sample acts as the carrier,while a fixed volume of reagent is Ž.injected Johnson and Petty,1982.All the funda-mental principles of flow injection analysis apply to rFIA,namely reproducible timing,injection,and dis-Ž.persion Ruzicka and Hansen,1988.rFIA allowsa Fig.1.Manifold diagram for the fluorescent,high-sensitivity sensor package for inorganic nitrogen nutrients.All flow rates are in ml r min.()R.T.Masserini Jr.,K.A.Fanning r Marine Chemistry682000323–333327background signal to be established prior to the injection of the reagent.Once a sample’s background fluorescence achieves a plateau,the reagent is in-jected to develop the fluorescent signal proportional to the nitrite present in the sample.In the case of seawater,the initial plateau height for a sample results from fluorescence due to naturally occurring DOM,the difference between the refractive index of the sample and the DIW wash between samples,and any contaminant organic matter from the tubing used. This plateau height,referred to as Background Fluo-Ž.rescence Level BFL,is subtracted from the overall sample fluorescence to correct for these interferences Ž.see Fig.2.In the final rFIA technique,DIW was used as the baseline into which the aniline reagent was injected in order to determine its contribution to the total fluorescence signal.Since DIW is very low in both DOM and nitrite,injection of the reagent solution produces peaks with no initial plateaus,and the heights of these peaks can be used as reagent blanks. The refractive index difference between the DIW wash and seawater samples contributed to the BFL Ž.slightly s3%.This was determined by analyzingŽ.0.68M NaCl99.999%Alfa Asear samples.The high purity NaCl was calcined to oxidize any impuri-ties and so was assumed to contain no nitrite or DOM.In a final step to avoid contamination,pump tubing made of platinum-cured silicon,a highly pure material that contains extremely low concentrations of leachable fluorescent contaminates,was used to carry all solutions within the analytical manifold. Besides reducing the degree of contamination by placticizers in the pump tubing,the baseline drift of the instrument was also greatly reduced.2.2.5.ReagentsThe aniline concentration in the aniline reagent solution was optimized to obtain the desired sensitiv-ity,while keeping the reagent-blank contribution toŽ.the fluorescence at a level see Fig.2that did not limit the dynamic range of the method.Flow rates and hydrogen ion concentration of an HCl reagent solution were selected to dilute the seawater sample enough that the background fluorescence did not limit the dynamic range of the method while still providing the optimum hydrogen ion concentration for the reaction.In the nitrate manifold an imidazole buffer was required.Its flow rate was minimized to limit the extent of dilution of the sample,and the imidazole concentration was increased until the free Cd metal ions were complexed sufficiently to prevent their forming a fluorescent complex with the aniline reagent.The total volume of the cadmium column was adjusted until99.9q%reduction efficiency was obtained.As a result of these tests,reagents for the fluores-cent rFIA nitrite and nitrate q nitrite methods are prepared as follows.DIW for both reagents and washing is given a final polishing with a Millipore Milli-Q RG system.The aniline reagent solution isŽ. composed of500m l of aniline Alfa Aseardiluted Fig.2.Peak record for fluorescent nitrite analysis.The first two peaks in the nitrite stripchart are reagent blanks.The next12peaks areŽ.duplicates of spiked Low Nutrient Seawater LNSW samples corresponding to0,413,207,103,41,and0nM nitrite,followed by duplicates of seawater samples.()R.T.Masserini Jr.,K.A.Fanning r Marine Chemistry682000323–333 328Ž.to1l with10%HCl V:V.The imidazole buffersolution for nitrate reduction consists of2.0g of Ž. imidazole reagent grade Aldrich Chemical plus15.0 ml of NH Cl r CuSO solution diluted to1l with 44DIW.The NH Cl r CuSO solution consists of250g44NH Cl plus2.5ml0.08M CuSO diluted to1l with 44DIW.Cadmium for nitrate reduction is prepared by washing coarse ground cadmium for reduction reac-Ž.tors CB r M Manufacturing with the following se-quence of solutions:acetone,DIW,10%HCl,DIW, imidazole buffer.The cadmium is then copperizedby immersion in0.08M CuSO.Supernatant liquid4is decanted and the cadmium is stored in the imida-zole buffer solution.The DIW wash and10%HCl reagent solutions are sparged with helium as de-scribed previously.2.3.Nitrate r nitrite analytical manifoldsThe lower half of Fig.1presents the analytical manifolds for fluorescent nitrite and nitrate q nitrite analyses.Solutions are delivered in Pt-cured silicon tubingŽ. by an Ismatec16channel peristaltic pump IPC-16 operated at25%of full speed.Flow rates are indi-cated in ml r min.Tubing downstream from the pump is0.8mm i.d.PTFE.A two-position,10-port Chem-inert injection valve with micro-electric actuator Ž.Valco Instrument C12-3116EH,equipped with twoŽ0.15ml sample loops no additional tubing between.ports2and5and10and7is used to introduce the aniline reagent into the reaction manifolds for both nitrite and nitrate plus nitrite.Upstream from the pump,an additional two-posi-tion,six-port Cheminert valve is configured as aŽ.sample-stream selection valve Fig.1.This valve shunts to waste the intersample air bubble that enters the system when the autosampler probe is between samples.During the shunting a DIW wash is se-lected.In the nitrite manifold,the first item after the pump is a tee where10%HCl reagent merges with the sample stream,which then flows to port4of the aniline-reagent injection valve.The acidified sample Ž.with or without aniline-reagent flows through portŽ3of this valve into400cm of PTFE tubing0.8mm .Ž. i.d.coiled inside a Technicon heater B-273-27setat708C.The heated nitrite analyte stream then leaves the heater and enters a5-cm section of high densityŽmicroporous PTFE tubing2mm i.d.,International.Polymer Engineering where any bubbles that have formed can escape to the atmosphere.From this tubing,the analyte stream flows to a fluorometer for determination of analyte concentration.The nitrate q nitrite reaction manifold begins after the pump with a merging of the imidazole buffer and the sample stream through a T-connection.Then comes4cm of PTFE tubing,followed by a908-Ž.flow-path,3-port PTFE valve Omnifit,followed by the cadmium column,followed by another3-port PTFE valve.The two3-port valves are for isolation of the cadmium column when the instrument is not being used to analyze samples.The cadmium columnŽ.is a polyvinylchloride PVC13.1-cm cylinder with aŽ1.272cm o.d.and a0.491cm i.d corresponding to a.bed-volume of approximately2.2ml packed withŽcoarse copperized cadmium powder MC r B Manu-.facturing prepared as described above.Four cm of PTFE tubing connects the second908flow-path valve to a second tee where the10%HCl reagent is merged.This tee connects directly to port8of the aniline-reagent injection valve.The acidified sample Ž.with or without aniline-reagent flows through port 9of this valve into400cm of the PTFE tubing in a 708C heater.Following the heater,the sample stream flows through5cm of high density microporous PTFE tubing to permit the escape of any bubbles formed during heating,and then to a fluorometer forŽdetection of the concentration of nitrite q nitrate as.additional nitrite.Although Fig.1shows separate heaters for the nitrite and nitrite q nitrate analyte streams,we use only one Technicon B-273-27heater which has been re-engineered to accept the two analyte streams in separate400cm long pieces of PTFE tubing.Both fluorometers into which the two analyte streams flow have Hitachi L-7480fluorescence de-tectors equipped with40-m l flowcells.Each detector is set up using the following parameters:220nm excitation,295nm emission,time constant s8s, PMT s1.Unknowns,blanks,and standards are introducedŽby linking an A.I.Scientific XYZ autosampler AIM .1250to the analytical manifold.Contact closure relays on the autosampler are used to signal the injection-valve actuators and sample-stream-selec-()R.T.Masserini Jr.,K.A.Fanning r Marine Chemistry682000323–333329tion-valve actuator and thereby synchronize the in-jections with the acquisition of aliquots of un-knowns,standard solutions,or DIW wash water.The synchronization is accomplished using the Concord software provided with the autosampler.At time equals0s for each aliquot,the reagent injection valves are set to the load position,and the sample-stream selection valve is set to the DIW wash posi-tion.At time equals60s the sample-stream selection valve is switched to the sample position;thus provid-ing a60-s wash period between aliquots.At time equals195s the reagent injection valve is switchedŽto the inject position the135-sec period between the beginning of the sample and the injection of the reagent allows for the sample background fluores-.cence to achieve a plateau,see Fig.2.After5s of reagent injection,the cycle is complete with a total elapsed time per sample of200s.This sequence of events causes the aniline reagent to fill and flush the injection loop during the first195s of the cycle. During the last5s of the cycle the sample stream sweeps the slug of aniline reagent into the reaction manifold.To prevent contamination of the seawater or aque-ous medium being analyzed during the sampling process,individual samples are quickly introduced into acid-washed,30-ml EPA screw-cap vials that are sealed with20mm PTFE r silicon septa.In order to extract aliquots from these sealed vials,the A.I.Ž.Scientific XYZ autosampler Fig.1is modified to use a stainless steel,12-in.,18-gauge luer-hub needle as a sample probe.An acrylic plate with precisely machined holes is placed over the tray holding the sample vials.The holes permit the probe to penetrate the vial,and the solid portions of the plate between the holes hold the vials in place when the probe is retracted.In order to maintain a consistent flow of sample through all manifolds the sealed vials must be vented during sampling.The venting mechanism consists of an aluminum block with two holes corre-sponding to the outer diameters of the18-gauge sampling needle and a shorter stainless steel22-gauge vent needle that also penetrates the septa on the vials.The vent-needle assembly is attached just be-low the autosampler probe and is held in place by setscrews.To compensate for the resistance of the silicon septa,a360-g stainless steel weight is at-tached to the end of the autosampler arm.All connections within the nitrate and nitrate q nitrite analytical manifolds are made with zero-dead-volume flange fittings with1r4-28thread con-nectors,tefzel tees,and barbed-to-1r4-28adapters Ž.for the calibrated autoanalysis pump tubing.2.4.Data acquisition and processingThe analog outputs of the fluorescence detectors are digitized with a24-bit DT2804A r D board installed in an AST docking station that is linked to an Ascentia950N90mHz Pentium laptop com-puter.The computer simultaneously controls the in-strument and collects and analyzes data.Signals are recorded and displayed in real time using Chromper-Ž.fect 2.1for Windows chromatography software Ž.Justice Innovations.The multi-tasking environment of Windows allows the autosampler to be controlled by the separate program,Concord2.8,provided with the autosampler.Digitized raw data for each channel are then imported into Peak Fit software4.1and processed via the Gaussian Deconvolution option. Peak-height determinations are exported to a spread-sheet where all corrections are applied and concen-trations are calculated.The corrections that are applied to the raw data are as follows.Peak heights of the unknown samples and standards are corrected for reagent blank fluores-cence by averaging the peak heights of two reagentŽ. injections into DIW which has no DOM and then subtracting the average from all peaks.Reagent-blank-corrected peak heights for unknowns and stan-dards are then further corrected for background fluo-rescence due to any DOM present and for the refrac-tive index differences of the wash and sample by subtracting the height of the plateau in the portion of the peak just ahead of the peak maximum producedŽby aniline-reagent injection see BFL in Fig.2,a.typical nitrite analysis peak record.These twice-cor-rected standards are then further corrected for ana-Ž.lyte nitrite and r or nitrate present in the solvent ŽLow Nutrient Seawater or LNSW used to match the.matrix of the seawater unknowns by subtracting the average peak height of two non-spiked LNSW sam-ples.The corrected standards’peaks are then used to calculate a standard curve,the slope of which is multiplied by the final corrected peak heights to obtain concentration values for actual samples()R.T.Masserini Jr.,K.A.Fanning r Marine Chemistry 682000323–333330Fig.3.Calibration curve for the fluorescent nitrite method.Stan-dards are spiked LNSW dilutions.Žsee Fig.3for the nitrite channel based on peaks in .Fig.2.A representative peak record for a standard curve for a nitrate dilution series can be seen in Fig.4.The Ž.slope for this analysis see Fig.5is approximately Ž.twice that for a nitrite curve Fig.3as a result of the greater dilution of the sample by the buffer and quenching effect imidazole has on the fluorescence of the reaction.2.5.Detection limitsTwenty DIW reagent blanks were measured and the detection limit defined as 2=the standarddevia-Fig.5.Calibration curve for the fluorescent nitrate q nitrite method.Standards are spiked LNSW dilutions.Ž.tion of the blanks Jones,1991.This yielded a detection limit for the nitrite channel of 4.6nM Ž.nitrite coefficient of variation:1.03%.A similar test of the nitrate q nitrite channel detection limit Žyielded a detection limit of 6.9nM coefficient of .variation:1.12%.2.6.Salinity effectsStandardizations were performed on three dilution series of nitrite at the following salinities:0.000,Ž.17.981,36.012see Fig.6.Each dilution series was run in duplicate.A regression of the slopes oftheFig.4.Peak record for fluorescent nitrate q nitrite analysis.The first two peaks in the nitrate stripchart are reagent blanks.The next six peaks are duplicates of spiked LNSW samples corresponding to 2002.5,1023.0and 0nM nitrate q nitrite.。
